Identification of Tumor-Associated Markers for Diagnosis and Therapy

ABSTRACT

The present technology relates to genetic products the expression of which is associated with cancer diseases. The present technology also relates to the therapy and diagnosis of diseases in which the genetic products are expressed or aberrantly expressed, in particular cancer diseases.

RELATED APPLICATIONS

The present application is a continuation of International PatentApplication No. PCT/EP08/08924, which was filed Oct. 22, 2008, claimingthe benefit of priority to European Patent Application No. 07020730.3,which was filed on Oct. 23, 2007. The entire text of the aforementionedapplications is incorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[Not Applicable]

BACKGROUND OF THE INVENTION

The present technology relates to nucleic acids and encoded polypeptideswhich are expressed in cancers. The present technology also relates toagents which bind the polypeptides. The nucleic acids, polypeptidescoded for by such nucleic acids and peptides derived therefrom, as wellas related antibodies and cytolytic T lymphocytes, are useful, interalia, in diagnostic and therapeutic contexts.

Despite interdisciplinary approaches and exhaustive use of classicaltherapeutic procedures, cancers are still among the leading causes ofdeath.

More recent therapeutic concepts in cancer therapy aim at incorporatingthe patient's immune system into the overall therapeutic concept byusing recombinant tumor vaccines and other specific measures such asantibody therapy. A prerequisite for the success of such a strategy isthe recognition of tumor-specific or tumor-associated antigens orepitopes by the patient's immune system whose effector functions are tobe interventionally enhanced.

Tumor cells biologically differ substantially from their nonmalignantcells of origin. These differences are due to genetic alterationsacquired during tumor development and result, inter alia, also in theformation of qualitatively or quantitatively altered molecularstructures in the cancer cells. Tumor-associated structures of this kindwhich are recognized by the specific immune system of thetumor-harboring host are referred to as tumor-associated antigens.

The specific recognition of tumor-associated antigens involves cellularand humoral mechanisms which are two functionally interconnected units:CD4⁺ and CD8⁺ T lymphocytes recognize the processed antigens presentedon the molecules of the MHC (major histocompatibility complex) classesII and I, respectively, while B lymphocytes produce circulating antibodymolecules which bind directly to unprocessed antigens. The potentialclinical-therapeutical importance of tumor-associated antigens resultsfrom the fact that the recognition of antigens on neoplastic cells bythe immune system leads to the initiation of cytotoxic effectormechanisms and, in the presence of T helper cells, can cause eliminationof the cancer cells (Pardon, Nat. Med. 4:525-31, 1998).

Antibody based cancer therapies have been successfully introduced intothe clinic and have emerged as the most promising therapeutics inoncology over the last decade. Eight antibodies have been approved fortreatment of neoplastic diseases, most of them, however in lymphoma andleukemia (Adams G P, Weiner L M, Nat Biotechnol 23:1147-57, 2005).

One of the challenges to be mastered for the advent of the nextgeneration of upgraded antibody-based cancer therapeutics is theselection of appropriate target molecules, which is the key for afavorable toxicity/efficacy profile.

The search for genes tightly silenced in the vast majority of healthytissues moves into the focus of attention the intriguing observationthat genes of the gametogenic and/or trophoblastic lineage arefrequently ectopically activated and robustly expressed in human cancer.Based on phenotypical similarities between germ cells, pregnancytrophoblast and cancer cells, John Beard proposed as much as 100 yearsago a “trophoblastic theory of cancer” (Beard J, Lancet 1:1758-63, 1902;Gurchot C, Oncology 31:310-3, 1975). The discovery of the sporadicproduction of chorionic gonadotropin, alpha-fetoprotein, CEA and othertrophoblastic hormones by cancer cells provided the first moleculesshared between neoplastic and trophoblastic cells (Acevedo H F et al.,Cancer 76:1467-75, 1995; Dirnhofer S et al., Hum Pathol 29:377-82, 1998;Gurchot C, Oncology 31:310-3, 1975; Iles R K, Chard T, J Urol 145:453-8,1991; Laurence D J, Neville A M, Br J Cancer 26:335-55, 1972). Theconcept was reignited by the inauguration of the steadily growingso-called cancer/germline (CG) class of genes, which represents morethan 100 members, each expressed in a variety of tumor types. Theobservation that entire trophoblastic and gametogenic programs escapetranscriptional silencing and are ectopically activated in cancer cells(Koslowski M et al., Cancer Res 64:5988-93, 2004; Simpson A J et al.,Nat Rev Cancer 5:615-25, 2005) indicates that within this class of geneswith exquisitely selective tissue distribution, appropriate targets formAB therapy may be found.

It was the object of the present technology to provide target structuresfor a diagnosis and therapy of cancers. This object is achieved by thesubject matter of the claims.

BRIEF SUMMARY OF THE INVENTION

According to the present technology, placenta-specific genes areidentified which are selectively or aberrantly expressed in tumor cellsand thus, provide target structures for therapeutic and diagnosticapproaches.

The nucleic acids identified according to the present technology to beselectively or aberrantly expressed in tumor cells are selected from thegroup consisting of (a) a nucleic acid which comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1-540, 541,545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591,595, 599, 602, 606, 610, 613, 617, 620, and 624 of the sequence listing,a part or derivative thereof, (b) a nucleic acid which hybridizes withthe nucleic acid of (a) under stringent conditions, (c) a nucleic acidwhich is degenerate with respect to the nucleic acid of (a) or (b), and(d) a nucleic acid which is complementary to the nucleic acid of (a),(b) or (c). These nucleic acids are also termed “tumor-associatednucleic acids” herein.

In another aspect, the present technology relates to antigens encoded bythe tumor-associated nucleic acids identified according to the presenttechnology. Accordingly, the tumor-associated antigens identifiedaccording to the present technology have an amino acid sequence encodedby a nucleic acid which is selected from the group consisting of (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1-540, 541, 545, 549, 553, 557, 560,563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606, 610,613, 617, 620, and 624 of the sequence listing, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c). In apreferred embodiment, the tumor-associated antigens identified accordingto the present technology comprise an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 542, 546, 550, 554, 567, 571, 584,588, 592, 596, 603, 607, 614, 621, and 625 of the sequence listing, apart or derivative thereof.

If, according to the present technology, reference is made to nucleicacids comprising certain nucleic acid sequences or tumor-associatedantigens comprising certain amino acid sequences this also includesembodiments wherein the nucleic acids or tumor-associated antigensconsist of these certain nucleic acid sequences or amino acid sequences,respectively.

The present technology generally relates to the use of tumor-associatednucleic acids and tumor-associated antigens identified according to thepresent technology or of parts or derivatives thereof, of nucleic acidsdirected against said tumor-associated nucleic acids, of antibodies or Tcells directed against the tumor-associated antigens identifiedaccording to the present technology or parts or derivatives thereofand/or of host cells expressing the tumor-associated antigens identifiedaccording to the present technology or parts or derivatives thereof fortherapy, prophylaxis, diagnosis and/or monitoring of neoplasticdiseases.

This may also involve the use of a combination of two or more of thesenucleic acids, antigens, antibodies, T cells and/or host cells.

In those embodiments of the present technology relating to the use ofantibodies directed against the tumor-associated antigens identifiedaccording to the present technology or parts or derivatives thereof alsoT cell receptors directed against the tumor-associated antigensidentified according to the present technology or parts or derivativesthereof, optionally in a complex with MHC molecules, may be used.

Especially suitable for therapy, prophylaxis, diagnosis and/ormonitoring is a part of the tumor-associated antigens identifiedaccording to the present technology which corresponds to thenon-transmembrane portion, in particular the extracellular portion ofthe tumor-associated antigens or is comprised thereof. Therefore,according to the present technology, a part of the tumor-associatedantigens identified according to the present technology whichcorresponds to the non-transmembrane portion, in particular theextracellular portion of the tumor-associated antigens or is comprisedthereof, or a corresponding part of the nucleic acids coding for thetumor-associated antigens identified according to the present technologyis preferred for therapy, prophylaxis, diagnosis and/or monitoring.Similarly the use of antibodies is preferred which are directed againsta part of the tumor-associated antigens identified according to thepresent technology which corresponds to the non-transmembrane portion,in particular the extracellular portion of the tumor-associated antigensor is comprised thereof.

Preferred diseases for a therapy, prophylaxis, diagnosis and/ormonitoring are those in which one or more of the tumor-associatednucleic acids identified according to the present technology areselectively expressed or abnormally expressed. Particularly preferreddiseases for a therapy, prophylaxis, diagnosis and/or monitoring arethose in which one or more of the tumor-associated nucleic acidsidentified according to the present technology and/or one or more of thetumor-associated antigens encoded thereby are selectively expressed orabnormally expressed.

In one aspect, the present technology relates to a pharmaceuticalcomposition comprising an agent which recognizes a tumor-associatedantigen identified according to the present technology or a nucleic acidcoding for the tumor-associated antigen and which is preferablyselective for cells which have expression or abnormal expression of atumor-associated antigen identified according to the present technology.

In a further aspect, the present technology relates to a pharmaceuticalcomposition comprising an agent which (I) inhibits expression oractivity of a tumor-associated antigen identified according to thepresent technology, and/or (II) has tumor-inhibiting or tumor-destroyingactivity and is selective for cells expressing or abnormally expressinga tumor-associated antigen identified according to the presenttechnology, and/or (III) when administered, selectively increases theamount of complexes between an MHC molecule and a tumor-associatedantigen identified according to the present technology or a partthereof, such as a peptide epitope. In particular embodiments, saidagent may cause induction of cell death, reduction in cell growth,damage to the cell membrane or secretion of cytokines and preferablyhave a tumor-inhibiting activity.

In one embodiment, the agent is an antisense nucleic acid whichhybridizes selectively with the nucleic acid coding for thetumor-associated antigen. In a further embodiment, the agent is a siRNApreferably comprising a sense RNA strand and an antisense RNA strand,wherein the sense and antisense RNA strands form an RNA duplex, andwherein the sense RNA strand comprises a nucleotide sequencesubstantially identical to a target sequence of about 19 to about 25contiguous nucleotides in a nucleic acid coding for the tumor-associatedantigen, preferably mRNA coding for the tumor-associated antigen. In afurther embodiment, the agent is an antibody which binds selectively tothe tumor-associated antigen, in particular a complement-activated ortoxin conjugated antibody which binds selectively to thetumor-associated antigen. In a preferred embodiment, the antibody whichbinds selectively to the tumor-associated antigen is coupled to atherapeutically useful substance and/or recruits natural or artificialeffector mechanisms to said cell expressing or abnormally expressingsaid tumor-associated antigen. In a further embodiment, the agent is acytotoxic T lymphocyte which recognizes the tumor-associated antigen ora part thereof bound by an MHC molecule on a cell and lyses the cellslabeled in this way. In a further embodiment, the agent is a T helperlymphocyte which recognizes the tumor-associated antigen or a partthereof bound by an MHC molecule on a cell and enhances effectorfunctions of other cells specifically recognizing said tumor-associatedantigen or a part thereof.

In a further embodiment, the agent comprises two or more agents whicheach recognize different tumor-associated antigens or different nucleicacids coding for tumor-associated antigens and/or inhibit expression oractivity of different tumor-associated antigens, and/or havetumor-inhibiting or tumor-destroying activity and are selective forcells expressing or abnormally expressing different tumor-associatedantigens, and/or when administered, selectively increase the amount ofcomplexes between MHC molecules and different tumor-associated antigensor parts thereof, wherein at least one of said differenttumor-associated antigens is a tumor-associated antigen identifiedaccording to the present technology.

Preferably, a tumor-associated antigen selectively limited to tumorsserves as a label for recruiting effector mechanisms to this specificlocation. In this aspect, the present technology includes embodimentswherein the agent itself does not have an ability to inhibit activity ofa tumor-associated antigen or a tumor-inhibiting or tumor-destroyingactivity but mediates such effect, in particular by recruiting effectormechanisms, in particular those having cell damaging potential, to aspecific location, in particular a tumor or tumor cells.

Preferably, said cells expressing or abnormally expressing atumor-associated antigen identified according to the present technologyare non-placenta cells.

The activity of a tumor-associated antigen identified according to thepresent technology can be any activity of a protein or a peptide. In oneembodiment this activity is an enzymatic activity.

According to the present technology the phrase “inhibit expression oractivity” includes a complete or essentially complete inhibition ofexpression or activity and a reduction in expression or activity.

The agent which, when administered, selectively increases the amount ofcomplexes between an MHC molecule and a tumor-associated antigenidentified according to the present technology or a part thereofcomprises one or more components selected from the group consisting of(i) the tumor-associated antigen or a part thereof, (ii) a nucleic acidwhich codes for said tumor-associated antigen or a part thereof, (iii) ahost cell which expresses said tumor-associated antigen or a partthereof, and (iv) isolated complexes between peptide epitopes from saidtumor-associated antigen and an MHC molecule.

The present technology furthermore relates to a pharmaceuticalcomposition which comprises one or more components selected from thegroup consisting of (i) a tumor-associated antigen identified accordingto the present technology or a part thereof, (ii) a nucleic acid whichcodes for a tumor-associated antigen identified according to the presenttechnology or a part thereof, (iii) an antibody which binds to atumor-associated antigen identified according to the present technologyor to a part thereof, (iv) an antisense nucleic acid which hybridizesspecifically with a tumor-associated nucleic acid identified accordingto the present technology/a nucleic acid coding for a tumor-associatedantigen identified according to the present technology, (v) an siRNAdirected against a tumor-associated nucleic acid identified according tothe present technology/a nucleic acid coding for a tumor-associatedantigen identified according to the present technology, (vi) a host cellwhich expresses a tumor-associated antigen identified according to thepresent technology or a part thereof, and (vii) isolated complexesbetween a tumor-associated antigen identified according to the presenttechnology or a part thereof and an MHC molecule.

In one embodiment, a nucleic acid coding for a tumor-associated antigenidentified according to the present technology or a part thereof ispresent in the pharmaceutical composition in an expression vector andfunctionally linked to a promoter. In a further embodiment, a nucleicacid coding for a tumor-associated antigen identified according to thepresent technology or a part thereof is present in the pharmaceuticalcomposition in a virus as further described below.

A host cell present in a pharmaceutical composition of the presenttechnology may secrete the tumor-associated antigen or the part thereof,may express it on the surface and preferably may additionally express anMHC molecule which binds to said tumor-associated antigen or said partthereof. In one embodiment, the host cell expresses the MHC moleculeendogenously. In a further embodiment, the host cell expresses the MHCmolecule and/or the tumor-associated antigen or the part thereof in arecombinant manner. The host cell is preferably nonproliferative. In apreferred embodiment, the host cell is an antigen-presenting cell, inparticular a dendritic cell, a monocyte or a macrophage.

In a further embodiment, an antibody present in a pharmaceuticalcomposition of the present technology is a monoclonal antibody. Infurther embodiments, the antibody is a chimeric or humanized antibody, afragment of an antibody or a synthetic antibody. The antibody may becoupled to a therapeutically or diagnostically useful agent also termedtherapeutic or diagnostic agent herein.

An antisense nucleic acid present in a pharmaceutical composition of thepresent technology may comprise a sequence of 6-50, in particular 10-30,15-30 and 20-30, contiguous nucleotides of the nucleic acid coding forthe tumor-associated antigen identified according to the presenttechnology.

In further embodiments, a tumor-associated antigen or a part thereof,provided by a pharmaceutical composition of the present technologyeither directly or via expression of a nucleic acid, binds to MHCmolecules on the surface of cells, said binding preferably causing acytolytic response and/or inducing cytokine release.

A pharmaceutical composition of the present technology may comprise apharmaceutically compatible carrier and/or an adjuvant.

A pharmaceutical composition of the present technology is preferablyused for the treatment or prevention of a disease characterized byselective expression or abnormal expression of a tumor-associatednucleic acid and/or tumor-associated antigen. In a preferred embodiment,the disease is a neoplastic disease, preferably cancer.

In a preferred embodiment, the pharmaceutical composition of the presenttechnology is in the form of a vaccine which may be used therapeuticallyor prophylactically. Such vaccine preferably comprises atumor-associated antigen identified according to the present technologyor a part thereof, and/or a nucleic acid which codes for atumor-associated antigen identified according to the present technologyor a part thereof. In particular embodiments, the nucleic acid ispresent in a virus or host cell.

The present technology furthermore relates to methods of treating,preventing, diagnosing or monitoring, i.e. determining the regression,progression, course and/or onset of, a disease characterized byexpression or abnormal expression of one of more tumor-associatednucleic acids identified according to the present technology, preferablyalso resulting in expression or abnormal expression of one of moretumor-associated antigens identified according to the presenttechnology, preferably a neoplastic disease, in particular cancer. Inone embodiment, the treatment or prevention comprises administering apharmaceutical composition of the present technology.

The methods of diagnosing and/or methods of monitoring according to thepresent technology generally concern the detection of and/ordetermination of the quantity of one or more parameters selected fromthe group consisting of (i) a tumor-associated nucleic acid identifiedaccording to the present technology, or a part thereof, (ii) atumor-associated antigen identified according to the present technology,or a part thereof, (iii) an antibody against a tumor-associated antigenidentified according to the present technology or a part thereof, and(iv) T lymphocytes, preferably cytotoxic or T helper lymphocytes, whichare specific for a tumor-associated antigen identified according to thepresent technology or a part thereof and/or a complex between thetumor-associated antigen or a part thereof and an MHC molecule, in abiological sample isolated from a patient, preferably from a patienthaving said disease, being suspected of having or falling ill with saiddisease or having a potential for said disease. Means for accomplishingsaid detection and/or determination of the quantity are described hereinand will be apparent to the skilled person.

Preferably, the presence of said nucleic acid or said part thereof, saidtumor-associated antigen or said part thereof, said antibody and/or saidT lymphocytes and/or a quantity of said nucleic acid or said partthereof, said tumor-associated antigen or said part thereof, saidantibody and/or said T lymphocytes which is increased compared to apatient without said disease is indicative for the presence of saiddisease or a potential for a development of said disease.

The methods of diagnosing and/or monitoring of the present technologyalso include embodiments wherein by detection or determination of thequantity of said nucleic acid or said part thereof, saidtumor-associated antigen or said part thereof, said antibody and/or saidT lymphocytes it is possible to assess and/or prognose the metastaticbehavior of said disease, wherein, preferably, the presence of saidnucleic acid or said part thereof, said tumor-associated antigen or saidpart thereof, said antibody and/or said T lymphocytes and/or a quantityof said nucleic acid or said part thereof, said tumor-associated antigenor said part thereof, said antibody and/or said T lymphocytes which isincreased compared to a patient without said disease or without ametastasis of said disease is indicative for a metastatic behavior ofsaid disease or a potential for a metastatic behavior of said disease.

In particular embodiments, said detection or determination of thequantity comprises (i) contacting a biological sample with an agentwhich binds specifically to said tumor-associated nucleic acid or saidpart thereof, to said tumor-associated antigen or said part thereof, tosaid antibody or to said T lymphocytes, and (ii) detecting the formationof or determining the amount of a complex between the agent and thenucleic acid or the part thereof, the tumor-associated antigen or thepart thereof, the antibody, or the T lymphocytes.

In one embodiment, the disease is characterized by expression orabnormal expression of two or more different tumor-associated nucleicacids preferably also resulting in expression or abnormal expression oftwo or more different tumor-associated antigens and a detection ordetermination of the quantity comprises a detection or determination ofthe quantity of two or more different tumor-associated nucleic acids orof parts thereof, of two or more different tumor-associated antigens orof parts thereof, of two or more antibodies binding to said two or moredifferent tumor-associated antigens or to parts thereof and/or of two ormore T lymphocytes specific for said two or more differenttumor-associated antigens or parts thereof, or complexes thereof withMHC molecules. In a further embodiment, the biological sample isolatedfrom the patient is compared to a comparable normal biological sample.

The methods of monitoring according to the present technology preferablycomprise a detection of and/or determination of the quantity of one ormore of the parameters mentioned above in a first sample at a firstpoint in time and in a further sample at a second point in time, whereinthe course of the disease is determined by comparing the two samples.

Preferably, a level of said nucleic acid or said part thereof, saidtumor-associated antigen or said part thereof, said antibody and/or saidT lymphocytes which is increased in a sample compared to a sample takenearlier from a patient indicates that the patient has developed or isabout to develop cancer and/or a metastasis of cancer and/or a relapseof cancer. Preferably, a level of said nucleic acid or said partthereof, said tumor-associated antigen or said part thereof, saidantibody and/or said T lymphocytes which is decreased in a samplecompared to a sample taken earlier from a patient indicates regressionof cancer and/or a metastasis of cancer in said patient and thus,preferably indicates a successful cancer therapy.

According to the present technology, detection of a nucleic acid or of apart thereof or determining the quantity of a nucleic acid or of a partthereof may be carried out using a oligo- or polynucleotide probe whichhybridizes specifically to said nucleic acid or said part thereof or maybe carried out by selective amplification of said nucleic acid or saidpart thereof, e.g. by means of PCR amplification. In one embodiment, theoligo- or polynucleotide probe comprises a sequence of 6-50, inparticular 10-30, 15-30 and 20-30, contiguous nucleotides of saidnucleic acid.

In particular embodiments, the tumor-associated antigen or the partthereof which is to be detected or the quantity of which is to bedetermined in the methods of the present technology is presentintracellularly, on the cell surface or in a complex with an MHCmolecule.

According to the present technology, detection of a tumor-associatedantigen or of a part thereof or determining the quantity of atumor-associated antigen or of a part thereof may be carried out usingan antibody binding specifically to said tumor-associated antigen orsaid part thereof.

According to the present technology, detection of an antibody ordetermining the quantity of an antibody may be carried out using aprotein or peptide binding specifically to said antibody.

According to the present technology, detection of or determining thequantity of T lymphocytes which are specific for a tumor-associatedantigen or a part thereof and/or a complex thereof with an MHC moleculemay be carried out using a cell presenting the complex between saidtumor-associated antigen or said part thereof and an MHC molecule. Tlymphocytes may additionally be detected by detecting theirproliferation, their cytokine production, and their cytotoxic activitytriggered by specific stimulation with a complex of an MHC molecule anda tumor-associated antigen or a part thereof. T lymphocytes may also bedetected with aid of a recombinant MHC molecule or a complex of two ormore MHC molecules loaded with immunogenic fragments of one or moretumor-associated antigens.

An agent which is used for detection or determining the quantity in themethods of the present technology such as a oligo- or polynucleotideprobe, an antibody, a protein or peptide or a cell is preferably labeledin a detectable manner, in particular by a detectable marker such as aradioactive marker or an enzymic marker.

In a particular aspect, the present technology relates to a method oftreating, preventing, diagnosing or monitoring a disease characterizedby expression or abnormal expression of a tumor-associated antigenidentified according to the present technology, which method comprisesadministering an antibody which binds to said tumor-associated antigenor to a part thereof and which is coupled to a therapeutic or diagnosticagent. The antibody may be a monoclonal antibody. In furtherembodiments, the antibody is a chimeric or humanized antibody or afragment of an antibody.

In certain embodiments, the methods of the present technology ofdiagnosing or monitoring a disease are performed with a biologicalsample containing or suspected of containing disseminating tumor cellsor metastatic tumor cells. Such biological samples include, for example,blood, serum, bone marrow, sputum, bronchial aspirate, and/or bronchiallavage. Preferably, the methods of the present technology of diagnosingor monitoring a disease are performed with a biological sample notcontaining placental cells and, in particular, being a non-placentabiological sample isolated from a subject.

In one particular aspect, the present technology relates to a method oftreating a patient having a disease characterized by expression orabnormal expression of a tumor-associated antigen identified accordingto the present technology, which method comprises (i) providing a samplecontaining immunoreactive cells, either obtained from said patient orfrom another individual of the same species, in particular a healthyindividual, or an individual of a different species, (ii) contactingsaid sample with a host cell expressing said tumor-associated antigen ora part thereof, under conditions which favor production of cytolytic Tcells against said tumor-associated antigen or a part thereof, and (iii)introducing the cytolytic T cells into the patient in an amount suitablefor lysing cells expressing the tumor-associated antigen or a partthereof. In one embodiment, the method includes cloning of the T cellreceptor of cytolytic T cells obtained and transferring the nucleic acidcoding for the T cell receptor to T cells, either obtained from saidpatient or from another individual of the same species, in particular ahealthy individual, or an individual of a different species, which Tcells thus receive the desired specificity and, as under (iii), may beintroduced into the patient.

In one embodiment, the host cell endogenously expresses an MHC molecule.In a further embodiment, the host cell recombinantly expresses an MHCmolecule and/or the tumor-associated antigen or the part thereof.Preferably, the host cell presents the tumor-associated antigen or thepart thereof by MHC molecules on its surface. The host cell ispreferably nonproliferative. In a preferred embodiment, the host cell isan antigen-presenting cell, in particular a dendritic cell, a monocyteor a macrophage.

The present technology also relates to a method of treating a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the present technology, which methodcomprises (i) identifying cells from the patient which express abnormalamounts of the tumor-associated antigen, (ii) isolating a sample of saidcells, (iii) culturing said cells, and (iv) introducing said cells intothe patient in an amount suitable for triggering an immune response tothe cells.

The present technology furthermore relates to a nucleic acid selectedfrom the group consisting of (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:1-540, 541, 545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583,587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and 624, a part orderivative thereof, (b) a nucleic acid which hybridizes with the nucleicacid of (a) under stringent conditions, (c) a nucleic acid which isdegenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c).

In a further aspect, the present technology relates to a recombinantnucleic acid molecule, in particular DNA or RNA molecule, whichcomprises a nucleic acid of the present technology.

The present technology also relates to host cells which contain anucleic acid or recombinant nucleic acid molecule of the presenttechnology.

The host cell may also comprise a nucleic acid coding for a MHCmolecule. In one embodiment, the host cell endogenously expresses theMHC molecule. In a further embodiment, the host cell recombinantlyexpresses the MHC molecule and/or the nucleic acid or recombinantnucleic acid molecule of the present technology or a part thereof.Preferably, the host cell is nonproliferative. In a preferredembodiment, the host cell is an antigen-presenting cell, in particular adendritic cell, a monocyte or a macrophage.

In a further embodiment, the present technology relates tooligonucleotides which hybridize with a nucleic acid identifiedaccording to the present technology and which may be used as geneticprobes or as “antisense” molecules. Nucleic acid molecules in the formof oligonucleotide primers or competent probes, which hybridize with anucleic acid identified according to the present technology or partsthereof, may be used for detecting said nucleic acid and/or findingnucleic acids which are homologous to said nucleic acid identifiedaccording to the present technology, e.g. by PCR amplification, Southernand Northern hybridization. Hybridization may be carried out under lowstringency, more preferably under medium stringency and most preferablyunder high stringency conditions.

In a further aspect, the present technology relates to a protein orpeptide which is encoded by a nucleic acid selected from the groupconsisting of (a) a nucleic acid which comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-540, 541, 545, 549,553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599,602, 606, 610, 613, 617, 620, and 624, a part or derivative thereof, (b)a nucleic acid which hybridizes with the nucleic acid of (a) understringent conditions, (c) a nucleic acid which is degenerate withrespect to the nucleic acid of (a) or (b), and (d) a nucleic acid whichis complementary to the nucleic acid of (a), (b) or (c). In a preferredembodiment, the protein or peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 542, 546, 550, 554,567, 571, 584, 588, 592, 596, 603, 607, 614, 621, and 625 of thesequence listing, a part or derivative thereof.

In a further aspect, the present technology relates to an immunogenicfragment of a tumor-associated antigen identified according to thepresent technology. Said fragment preferably binds to a MHC molecule oran antibody, preferably to a human HLA receptor or a human antibody.According to the present technology, a part or fragment preferablycomprises a sequence of at least 5, at least 6, in particular at least8, at least 10, at least 12, at least 15, at least 20, at least 30 or atleast 50, amino acids.

In a further aspect, the present technology relates to an agent whichbinds to a tumor-associated antigen identified according to the presenttechnology or to a part thereof. In a preferred embodiment, the agent isa protein or peptide, in particular an antibody, a T cell receptor or anMHC molecule. In further embodiments, the antibody is a monoclonal,chimeric, or humanized antibody, an antibody produced by combinatorytechniques, or a fragment of an antibody. In one preferred embodiment,the present technology relates to an antibody which binds selectively toa complex of (i) a tumor-associated antigen identified according to thepresent technology or a part thereof and (ii) an MHC molecule to whichsaid tumor-associated antigen identified according to the presenttechnology or said part thereof binds, with said antibody not binding to(i) or (ii) alone.

According to the present technology, the term “binding” preferablyrelates to a specific binding. “Specific binding” means that an agentsuch as an antibody binds stronger to a target such as an epitope forwhich it is specific compared to the binding to another target. An agentbinds stronger to a first target compared to a second target if it bindsto the first target with a dissociation constant (K_(D)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant (K_(D)) for the target to which the agent bindsspecifically is more than 10-fold, preferably more than 20-fold, morepreferably more than 50-fold, even more preferably more than 100-fold,200-fold, 500-fold or 1000-fold lower than the dissociation constant(K_(D)) for the target to which the agent does not bind specifically.

Such specific antibodies may, for example, be obtained by immunizationusing the aforementioned peptides.

The present technology furthermore relates to a conjugate between anagent of the present technology which binds to a tumor-associatedantigen identified according to the present technology or to a partthereof or an antibody of the present technology and a therapeutic ordiagnostic agent. In one embodiment, the therapeutic or diagnostic agentis a toxin.

In a further aspect, the present technology relates to a kit fordetecting a disease characterized by expression or abnormal expressionof one of more tumor-associated nucleic acids identified according tothe present technology, preferably also resulting in expression orabnormal expression of one of more tumor-associated antigens identifiedaccording to the present technology, preferably a neoplastic disease, inparticular cancer, which kit comprises agents for detection ordetermining the quantity (i) of the tumor-associated nucleic acid or ofa part thereof, (ii) of the tumor-associated antigen or of a partthereof, (iii) of antibodies which bind to the tumor-associated antigenor to a part thereof, and/or (iv) of T cells which are specific for thetumor-associated antigen or a part thereof or a complex thereof with anMHC molecule. Such agents are described herein above.

In one embodiment, the present technology relates to a pharmaceuticalcomposition which comprises an agent that (I) inhibits expression oractivity of a tumor-associated antigen and/or (II) has tumor-inhibitingactivity, and is selective for cells expressing or abnormally expressinga tumor-associated antigen and/or (III) when administered, selectivelyincreases the amount of complexes between an MHC molecule and atumor-associated antigen or a part thereof, the tumor-associated antigenhaving a sequence encoded by a nucleic acid which is selected from thegroup consisting of: (a) a nucleic acid which comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 541, 1-540,545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591,595, 599, 602, 606, 610, 613, 617, 620, and 624, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c).

In another embodiment, the present technology relates to apharmaceutical composition which comprises one or more componentsselected from the group consisting of: (i) a tumor-associated antigen ora part thereof, (ii) a nucleic acid which codes for a tumor-associatedantigen or a part thereof, (iii) an antibody which binds to atumor-associated antigen or a part thereof, (iv) an antisense nucleicacid which hybridizes specifically with a nucleic acid coding for atumor-associated antigen, (v) an siRNA directed against a nucleic acidcoding for a tumor-associated antigen, (vi) a host cell which expressesa tumor-associated antigen or a part thereof, and (vii) isolatedcomplexes between a tumor-associated antigen or a part thereof and anMHC molecule, said tumor-associated antigen having a sequence encoded bya nucleic acid which is selected from the group consisting of: (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560,563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606, 610,613, 617, 620, and 624, a part or derivative thereof, (b) a nucleic acidwhich hybridizes with the nucleic acid of (a) under stringentconditions, (c) a nucleic acid which is degenerate with respect to thenucleic acid of (a) or (b), and (d) a nucleic acid which iscomplementary to the nucleic acid of (a), (b) or (c).

In yet another embodiment, the present technology relates to a method ofdiagnosing or monitoring a cancer disease which comprises detecting ordetermining the quantity (i) of a tumor-associated nucleic acid or of apart thereof, and/or (ii) of a tumor-associated antigen or of a partthereof, and/or (iii) of an antibody to the tumor-associated antigen ora part thereof and/or (iv) of T lymphocytes which are specific to thetumor-associated antigen or to a part thereof in a biological sampleisolated from a patient, said tumor-associated nucleic acid beingselected from the group consisting of: (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574,577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and624, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c), and said tumor-associated antigen having a sequenceencoded by a nucleic acid which is selected from said group of nucleicacids.

In a further embodiment, the present technology relates to a method oftreating or preventing a disease characterized by expression or abnormalexpression of a tumor-associated antigen which comprises administrationof a pharmaceutical composition of the present technology, saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from the group consisting of: (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574,577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and624, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c).

In yet another embodiment, the present technology relates to a method oftreating, preventing, diagnosing or monitoring a disease characterizedby expression or abnormal expression of a tumor-associated antigen whichcomprises administering an antibody that binds to said tumor-associatedantigen or to a part thereof and is coupled to a therapeutic ordiagnostic agent, said tumor-associated antigen having a sequenceencoded by a nucleic acid which is selected from the group consistingof: (a) a nucleic acid which comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 541, 1-540, 545, 549, 553, 557,560, 563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606,610, 613, 617, 620, and 624, a part or derivative thereof, (b) a nucleicacid which hybridizes with the nucleic acid of (a) under stringentconditions, (c) a nucleic acid which is degenerate with respect to thenucleic acid of (a) or (b), and (d) a nucleic acid which iscomplementary to the nucleic acid of (a), (b) or (c).

Another embodiment of the present technology relates to a method oftreating a patient having a disease characterized by expression orabnormal expression of a tumor-associated antigen which comprises: (i)providing a sample containing immunoreactive cells, (ii) contacting saidsample with a host cell expressing said tumor-associated antigen or apart thereof, under conditions which favor production of cytolytic orcytokine-releasing T cells against said tumor-associated antigen or saidpart thereof, and (iii) introducing the cytolytic or cytokine-releasingT cells into the patient in an amount suitable for lysing cellsexpressing the tumor-associated antigen or a part thereof, saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from the group consisting of: (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574,577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and624, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c).

An additional embodiment of the present technology relates to a methodof inhibiting the development of cancer in a patient which comprisesadministering an effective amount of a pharmaceutical composition of thepresent technology.

In yet another embodiment, the present technology relates to an agent,which binds specifically to a protein or polypeptide or to a partthereof, said protein or polypeptide being encoded by a nucleic acidselected from the group consisting of: (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574,577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and624, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c).

In an additional embodiment, the present technology relates to anantibody, which binds selectively to a complex of: (i) a protein orpolypeptide or a part thereof and (ii) an MHC molecule to which saidprotein or polypeptide or said part thereof binds, with said antibodynot binding to (i) or (ii) alone and said protein or polypeptide beingencoded by a nucleic acid selected from the group consisting of: (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560,563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606, 610,613, 617, 620, and 624, a part or derivative thereof, (b) a nucleic acidwhich hybridizes with the nucleic acid of (a) under stringentconditions, (c) a nucleic acid which is degenerate with respect to thenucleic acid of (a) or (b), and (d) a nucleic acid which iscomplementary to the nucleic acid of (a), (b) or (c).

In yet another embodiment, the present technology relates to a kit fordetecting cancer, which comprises agents for detecting or determiningthe quantity of (i) of a tumor-associated nucleic acid or of a partthereof, and/or (ii) of a tumor-associated antigen or of a part thereof,and/or (iii) of antibodies which bind to the tumor-associated antigen orto a part thereof, and/or (iv) of T cells which are specific for acomplex between the tumor-associated antigen or a part thereof and anMHC molecule, said tumor-associated nucleic acid being selected from thegroup consisting of: (a) a nucleic acid which comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 541, 1-540,545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591,595, 599, 602, 606, 610, 613, 617, 620, and 624, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c), and saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from said group of nucleic acids.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Expression of a tumor-associated nucleic acid identifiedaccording to the present technology in normal tissues and cancer tissue.Significant expression of the nucleic acid sequence according to SEQ IDNO:540 was found only in placenta tissue and mamma carcinomas.

FIG. 2. Quantitative expression of a tumor-associated nucleic acididentified according to the present technology in normal tissues andcancer tissue. Quantitative RT-PCR showed selective expression of thenucleic acid sequence according to SEQ ID NO:540 in placenta tissue andmamma carcinomas.

FIG. 3. Quantitative expression of SEQ ID NO:540 mRNA in MCF-7 breastcancer cells. Real-time RT-PCR 24 h after transfection with siRNA oligosshowed that both SEQ ID NO:540-specific siRNAs (siRNA #1 (SEQ ID NO:630,631), siRNA #2 (SEQ ID NO:632, 633)) induce robust silencing of SEQ IDNO:540 expression.

FIG. 4. Silencing of SEQ ID NO:540 expression by transfection with siRNAoligos results in impaired proliferation of MCF-7 breast cancer cells.Proliferation was quantified 96 h after transfection with siRNAs bymeasuring incorporation of BrdU in newly synthesized DNA strands. Theseresults show that SEQ ID NO:540 is a positive factor for theproliferation of breast cancer cells.

FIG. 5. Quantitative expression of SEQ ID NO:541 in normal tissues andcancer tissue. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:541 in lung cancer.

FIG. 6. Quantitative expression of SEQ ID NO:545 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:545 in malignant melanomas.

FIG. 7. Quantitative expression of SEQ ID NO:549 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:549 in ovarian cancer.

FIG. 8. Quantitative expression of SEQ ID NO:553 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:553 in colon cancer and ovariancancer.

FIG. 9. Quantitative expression of SEQ ID NO:557 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:557 in breast cancer.

FIG. 10. Quantitative expression of SEQ ID NO:560 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:560 in colon cancer and ovariancancer.

FIG. 11. Quantitative expression of SEQ ID NO:563 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:563 in breast cancer, colon cancer,ovarian cancer, lung cancer and melanoma.

FIG. 12. Quantitative expression of SEQ ID NO:566 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:566 in gastric cancer, breastcancer, colon cancer, ovarian cancer, lung cancer and melanoma.

FIG. 13. Quantitative expression of SEQ ID NO:570 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:570 in ovarian cancer, lung cancerand melanoma.

FIG. 14. Quantitative expression of SEQ ID NO:574 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:574 in lung cancer and melanoma.

FIG. 15. Quantitative expression of SEQ ID NO:577 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:577 in gastric cancer, breastcancer and lung cancer.

FIG. 16. Quantitative expression of SEQ ID NO:580 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:580 in ovarian cancer and lungcancer.

FIG. 17. Quantitative expression of SEQ ID NO:583 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:583 in colon cancer, ovarian cancerand lung cancer.

FIG. 18. Quantitative expression of SEQ ID NO:587 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:587 in lung cancer.

FIG. 19. Quantitative expression of SEQ ID NO:591 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:591 in breast cancer, colon cancer,ovarian cancer, lung cancer and melanoma.

FIG. 20. Quantitative expression of SEQ ID NO:595 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:595 in gastric cancer, coloncancer, ovarian cancer, lung cancer and melanoma.

FIG. 21. Quantitative expression of SEQ ID NO:599 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:599 in gastric cancer, breastcancer, lung cancer and melanoma.

FIG. 22. Quantitative expression of SEQ ID NO:602 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:602 in ovarian cancer and lungcancer.

FIG. 23. Quantitative expression of SEQ ID NO:606 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:606 in gastric cancer, colon cancerand lung cancer.

FIG. 24. Quantitative expression of SEQ ID NO:610 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:610 in gastric cancer, breastcancer and lung cancer.

FIG. 25. Quantitative expression of SEQ ID NO:613 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:613 in breast cancer, lung cancerand melanoma.

FIG. 26. Quantitative expression of SEQ ID NO:617 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:617 in lung cancer and melanoma.

FIG. 27. Quantitative expression of SEQ ID NO:620 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:620 in ovarian cancer and melanoma.

FIG. 28. Quantitative expression of SEQ ID NO:624 in normal tissues andcancer tissues. Real-time RT-PCR showed overexpression of the nucleicacid sequence according to SEQ ID NO:624 in gastric cancer and lungcancer.

DETAILED DESCRIPTION OF THE INVENTION

A reference herein to a range of numerical values is to be understood soas to specify and mention each of the individual numerical valuescomprised by said range. For example, a reference to SEQ ID NOs: 1-540is to be understood so as to refer to each and every of the followingindividual SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509,510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523,524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,538, 539, and 540.

According to the present technology, a “reference” such as a referencesample or reference organism may be used to correlate and compare theresults obtained in the methods of the present technology from a testsample or test organism, i.e. a patient. Typically the referenceorganism is a healthy organism, in particular an organism which does notsuffer from cancer.

A “reference value” can be determined from a reference empirically bymeasuring a sufficiently large number of references. Preferably thereference value is determined by measuring at least 2, preferably atleast 3, preferably at least 5, preferably at least 8, preferably atleast 12, preferably at least 20, preferably at least 30, preferably atleast 50, or preferably at least 100 references.

According to the present technology, a nucleic acid is preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidscomprise according to the present technology genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the present technology, a nucleic acid may be present as asingle-stranded or double-stranded and linear or covalently circularlyclosed molecule.

The terms “tumor-associated nucleic acid identified according to thepresent technology” and “nucleic acid encoding a tumor-associatedantigen identified according to the present technology” have similarmeanings. However, the different terms are used herein to account forthe fact that in some embodiments only the expression of nucleic acid,in particular mRNA, is of relevance while the expression of protein isnot a critical factor.

As used herein, the term “RNA” means a molecule comprising at least oneribonucleotide residue. By “ribonucleotide” is meant a nucleotide with ahydroxyl group at the 2′-position of a beta-D-ribo-furanose moiety. Theterm includes double stranded RNA, single stranded RNA, isolated RNAsuch as partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA, as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

If reference is made herein to the detection of or the determination ofthe quantity of a nucleic acid, the nucleic acid which is actually to bedetected or the quantity of which is actually to be determined ispreferably mRNA. However, it should be understood that this may alsoinclude embodiments wherein mRNA is detected or the quantity of mRNA isdetermined indirectly. For example, mRNA may be transformed into cDNAand the cDNA detected or its quantity determined. mRNA is given hereinas the cDNA equivalent. One skilled in the art would understand that thecDNA sequence is equivalent to the mRNA sequence, and can be used forthe same purpose herein, e.g., the generation of probes hybridizing tothe nucleic acid to be detected. Thus, if reference is made herein tothe sequences shown in the sequence listing this is also to include theRNA equivalents of said sequences.

The nucleic acids described according to the present technology havepreferably been isolated. The term “isolated nucleic acid” meansaccording to the present technology that the nucleic acid was (i)amplified in vitro, for example by polymerase chain reaction (PCR), (ii)recombinantly produced by cloning, (iii) purified, for example bycleavage and gel-electrophoretic fractionation, or (iv) synthesized, forexample by chemical synthesis. An isolated nucleic acid is a nucleicacid which is available for manipulation by recombinant DNA techniques.

A degenerate nucleic acid according to the present technology is anucleic acid that differs from a reference nucleic acid in codonsequence due to the degeneracy of the genetic code.

“Derivative” of a nucleic acid means according to the present technologythat single or multiple such as at least 2, at least 4, or at least 6and preferably up to 3, up to 4, up to 5, up to 6, up to 10, up to 15,or up to 20 nucleotide substitutions, deletions and/or additions arepresent in said nucleic acid. Furthermore, the term “derivative” alsocomprises chemical derivatization of a nucleic acid on a nucleotidebase, on the sugar or on the phosphate. The term “derivative” alsocomprises nucleic acids which contain nucleotides and nucleotide analogsnot occurring naturally.

Preferably the degree of identity between a specific nucleic acidsequence described herein and a nucleic acid sequence which is aderivative of said specific nucleic acid sequence, which hybridizes withsaid specific nucleic acid sequence and/or which is degenerate withrespect to said specific nucleic acid sequence will be at least 70%,preferably at least 75%, preferably at least 80%, more preferably atleast 85%, even more preferably at least 90% or most preferably at least95%, 96%, 97%, 98% or 99%. The degree of identity is preferably givenfor a region of at least about 30, at least about 50, at least about 70,at least about 90, at least about 100, at least about 150, at leastabout 200, at least about 250, at least about 300, or at least about 400nucleotides. In preferred embodiments, the degree of identity is givenfor the entire length of the reference nucleic acid sequence, such asthe nucleic acid sequences given in the sequence listing.

A nucleic acid is “complementary” to another nucleic acid if the twosequences are capable of hybridizing and forming a stable duplex withone another, with hybridization preferably being carried out underconditions which allow specific hybridization between polynucleotides(stringent conditions). Stringent conditions are described, for example,in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel etal., Editors, John Wiley & Sons, Inc., New York and refer, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15M sodium citrate, pH 7. After hybridization, the membrane to which theDNA has been transferred is washed, for example, in 2×SSC at roomtemperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68°C.

A percent complementarity indicates the percentage of contiguousresidues in a nucleic acid molecule that can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” or “fully complementary” meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. Preferably, the degree of complementarityaccording to the present technology is at least 70%, preferably at least75%, preferably at least 80%, more preferably at least 85%, even morepreferably at least 90% or most preferably at least 95%, 96%, 97%, 98%or 99%.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two polypeptide or nucleicacid sequences indicates the percentage of amino acids or nucleotidesthat are identical between the sequences.

The term “percentage identity” is intended to denote a percentage ofnucleotides or of amino acid residues which are identical between thetwo sequences to be compared, obtained after the best alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly and over their entire length.Sequence comparisons between two nucleotide or amino acid sequences areconventionally carried out by comparing these sequences after havingaligned them optimally, said comparison being carried out by segment orby “window of comparison” in order to identify and compare local regionsof sequence similarity. The optimal alignment of the sequences forcomparison may be produced, besides manually, by means of the localhomology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482,by means of the local homology algorithm of Neddleman and Wunsch, 1970,J. Mol. Biol. 48, 443, by means of the similarity search method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or bymeans of computer programs which use these algorithms (GAP, BESTFIT,FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

In one embodiment, a nucleic acid sequence which is a derivative of aspecific nucleic acid sequence, which is degenerate with respect to aspecific nucleic acid sequence or which is a part of a specific nucleicacid sequence has a relevant function and/or activity of the specificnucleic acid sequence, i.e. it may encode a protein or peptide havingthe same activity or immunological properties as the protein or peptideencoded by the specific nucleic acid sequence and, in one embodiment,encodes the same protein or peptide.

Nucleic acids coding for tumor-associated antigens may, according to thepresent technology, be present alone or in combination with othernucleic acids, in particular heterologous nucleic acids. In preferredembodiments, a nucleic acid is functionally linked to expression controlsequences or regulatory sequences which may be homologous orheterologous with respect to said nucleic acid. A coding sequence and aregulatory sequence are “functionally” linked to one another, if theyare covalently linked to one another in such a way that expression ortranscription of said coding sequence is under the control or under theinfluence of said regulatory sequence. If the coding sequence is to betranslated into a functional protein, then, with a regulatory sequencefunctionally linked to said coding sequence, induction of saidregulatory sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or said codingsequence not being capable of being translated into the desired proteinor peptide.

The term “expression control sequence” or “regulatory sequence”comprises according to the present technology promoters, enhancers andother control elements which regulate expression of a gene. Inparticular embodiments of the present technology, the expression controlsequences can be regulated. The exact structure of regulatory sequencesmay vary as a function of the species or cell type, but generallycomprises 5′untranscribed and 5′untranslated sequences which areinvolved in initiation of transcription and translation, respectively,such as TATA box, capping sequence, CAAT sequence, and the like. Morespecifically, 5′untranscribed regulatory sequences comprise a promoterregion which includes a promoter sequence for transcriptional control ofthe functionally linked gene. Regulatory sequences may also compriseenhancer sequences or upstream activator sequences.

According to the present technology, a nucleic acid may furthermore bepresent in combination with another nucleic acid which codes for apeptide controlling secretion of the protein or peptide encoded by saidnucleic acid from a host cell. According to the present technology, anucleic acid may also be present in combination with another nucleicacid which codes for a peptide causing the encoded protein or peptide tobe anchored on the cell membrane of the host cell or compartmentalizedinto particular organelles of said cell. Similarly, a combination with anucleic acid is possible which represents a reporter gene or any “tag”.

In a preferred embodiment, a recombinant nucleic acid molecule isaccording to the present technology a vector, where appropriate with apromoter, which controls expression of a nucleic acid, for example anucleic acid coding for a tumor-associated antigen identified accordingto the present technology. The term “vector” is used here in its mostgeneral meaning and comprises any intermediary vehicle for a nucleicacid which enables said nucleic acid, for example, to be introduced intoprokaryotic and/or eukaryotic cells and, where appropriate, to beintegrated into a genome. Vectors of this kind are preferably replicatedand/or expressed in the cells. An intermediary vehicle may be adapted,for example, to the use in electroporation, in bombardment withmicroprojectiles, in liposomal administration, in the transfer with theaid of agrobacteria or in insertion via DNA or RNA viruses. Vectorscomprise plasmids, phagemids, bacteriophages or viral genomes.

The nucleic acids coding for a tumor-associated antigen identifiedaccording to the present technology may be used for transfection of hostcells. Nucleic acids here mean both recombinant DNA and RNA. RecombinantRNA may be prepared by in-vitro transcription of a DNA template.Furthermore, it may be modified by stabilizing sequences, capping andpolyadenylation prior to application.

According to the present technology, the term “host cell” relates to anycell which can be transformed or transfected with an exogenous nucleicacid. The term “host cells” comprises according to the presenttechnology prokaryotic (e.g. E. coli) or eukaryotic cells (e.g.dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast cellsand insect cells). Particular preference is given to mammalian cellssuch as cells from humans, mice, hamsters, pigs, goats, primates. Thecells may be derived from a multiplicity of tissue types and compriseprimary cells and cell lines. Specific examples comprise keratinocytes,peripheral blood leukocytes, stem cells of the bone marrow and embryonicstem cells. In further embodiments, the host cell is anantigen-presenting cell, in particular a dendritic cell, monocyte or amacrophage. A nucleic acid may be present in the host cell in the formof a single copy or of two or more copies and, in one embodiment, isexpressed in the host cell.

According to the present technology, the term “expression” is used inits most general meaning and comprises the production of RNA or of RNAand protein. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.Preferred expression systems in mammalian cells comprise pcDNA3.1 andpRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selectablemarker such as a gene imparting resistance to G418 (and thus enablingstably transfected cell lines to be selected) and the enhancer-promotersequences of cytomegalovirus (CMV).

In those cases of the present technology in which a MHC moleculepresents a tumor-associated antigen or a part thereof, an expressionvector may also comprise a nucleic acid sequence coding for said MHCmolecule. The nucleic acid sequence coding for the MHC molecule may bepresent on the same expression vector as the nucleic acid coding for thetumor-associated antigen or the part thereof, or both nucleic acids maybe present on different expression vectors. In the latter case, the twoexpression vectors may be cotransfected into a cell. If a host cellexpresses neither the tumor-associated antigen or the part thereof northe MHC molecule, both nucleic acids coding therefor may be transfectedinto the cell either on the same expression vector or on differentexpression vectors. If the cell already expresses the MHC molecule, onlythe nucleic acid sequence coding for the tumor-associated antigen or thepart thereof can be transfected into the cell.

The present technology also comprises kits for detection and/ordetermination of the quantity of nucleic acids. Such kits comprise, forexample, a pair of amplification primers which hybridize to the nucleicacid which is to be detected or the amount of which is to be determined.The primers preferably comprise a sequence of 6-50, in particular 10-30,15-30 and 20-30 contiguous nucleotides of the nucleic acid and arenonoverlapping, in order to avoid the formation of primer dimers. One ofthe primers will hybridize to one strand of the nucleic acid, and theother primer will hybridize to the complementary strand in anarrangement which allows amplification of the nucleic acid.

“Antisense molecules” or “antisense nucleic acids” may be used forregulating, in particular reducing, expression of a nucleic acid. Theterm “antisense molecule” or “antisense nucleic acid” refers accordingto the present technology to an oligonucleotide which is anoligoribonucleotide, oligodeoxyribonucleotide, modifiedoligoribonucleotide or modified oligodeoxyribonucleotide and whichhybridizes under physiological conditions to DNA comprising a particulargene or to mRNA of said gene, thereby inhibiting transcription of saidgene and/or translation of said mRNA. According to the presenttechnology, an “antisense molecule” also comprises a construct whichcontains a nucleic acid or a part thereof in reverse orientation withrespect to its natural promoter. An antisense transcript of a nucleicacid or of a part thereof may form a duplex with naturally occurringmRNA and thus prevent accumulation of or translation of the mRNA.Another possibility is the use of ribozymes for inactivating a nucleicacid.

Antisense oligonucleotides preferred according to the present technologyhave a sequence of 6-50, in particular 10-30, 15-30 and 20-30,contiguous nucleotides of the target nucleic acid and preferably arefully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes withan N-terminal or 5′ upstream site such as a translation initiation site,transcription initiation site or promoter site. In further embodiments,the antisense oligonucleotide hybridizes with a 3′untranslated region ormRNA splicing site.

In one embodiment, an oligonucleotide of the present technology consistsof ribonucleotides, deoxyribonucleotides or a combination thereof, withthe 5′ end of one nucleotide and the 3′ end of another nucleotide beinglinked to one another by a phosphodiester bond. These oligonucleotidesmay be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the present technologyis a “modified” oligonucleotide. Here, the oligonucleotide may bemodified in very different ways, without impairing its ability to bindits target, in order to increase, for example, its stability ortherapeutic efficacy. According to the present technology, the term“modified oligonucleotide” means an oligonucleotide in which (i) atleast two of its nucleotides are linked to one another by a syntheticinternucleoside bond (i.e. an internucleoside bond which is not aphosphodiester bond) and/or (ii) a chemical group which is usually notfound in nucleic acids is covalently linked to the oligonucleotide.Preferred synthetic internucleoside bonds are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphatetriesters, acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotideshaving a covalently modified base and/or sugar. “Modifiedoligonucleotides” comprise, for example, oligonucleotides with sugarresidues which are covalently bound to low molecular weight organicgroups other than a hydroxyl group at the 3′ position and a phosphategroup at the 5′ position. Modified oligonucleotides may comprise, forexample, a 2′-O-alkylated ribose residue or another sugar instead ofribose, such as arabinose.

It is to be understood that all embodiments described above with respectto oligonucleotides may also apply to polynucleotides.

By “small interfering RNA” or “siRNA” as used herein is meant anisolated RNA molecule, preferably greater than 10 nucleotides in length,more preferably greater than 15 nucleotides in length, and mostpreferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in length that is used to identify a target gene or mRNA tobe degraded. A range of 19-25 nucleotides is the most preferred size forsiRNAs.

siRNA according to the present technology can comprise partiallypurified RNA, substantially pure RNA, synthetic RNA, or recombinantlyproduced RNA, as well as altered RNA that differs fromnaturally-occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the siRNAor to one or more internal nucleotides of the siRNA; modifications thatmake the siRNA resistant to nuclease digestion (e.g., the use of2′-substituted ribonucleotides or modifications to the sugar-phosphatebackbone); or the substitution of one or more nucleotides in the siRNAwith deoxyribonucleotides. Furthermore, siRNA may be modified toincrease the stability thereof as described above for modifiedoligonucleotides, in particular by introducing one or morephosphorothioate linkages.

One or both strands of the siRNA can also comprise a 3′-overhang. Asused herein, a “3′-overhang” refers to at least one unpaired nucleotideextending from the 3′-end of an RNA strand. Thus in one embodiment, thesiRNA comprises at least one 3′-overhang of from 1 to about 6nucleotides (which includes ribonucleotides or deoxynucleotides) inlength, preferably from 1 to about 5 nucleotides in length, morepreferably from 1 to about 4 nucleotides in length, and particularlypreferably from about 2 to about 4 nucleotides in length. In theembodiment in which both strands of the siRNA molecule comprise a3′-overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′-overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA of the present technology cancomprise 3′-overhangs of dideoxythymidylic acid (“TT”) or diuridylicacid (“uu”).

In order to enhance the stability of the siRNA, the 3′-overhangs can bealso stabilized against degradation. In one embodiment, the overhangsare stabilized by including purine nucleotides, such as adenosine orguanosine nucleotides. Alternatively, substitution of pyrimidinenucleotides by modified analogues, e.g., substitution of uridinenucleotides in the 3′-overhangs with 2′-deoxythymidine, is tolerated anddoes not affect the efficiency of RNAi degradation. In particular, theabsence of a 2′-hydroxyl in the 2′-deoxythymidine significantly enhancesthe nuclease resistance of the 3′-overhang in tissue culture medium.

The sense and antisense strands of the siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. That is, thesense region and antisense region can be covalently connected via alinker molecule. The linker molecule can be a polynucleotide ornon-nucleotide linker. Without wishing to be bound by any theory, it isbelieved that the hairpin area of the latter type of siRNA molecule iscleaved intracellularly by the “Dicer” protein (or its equivalent) toform a siRNA of two individual base-paired RNA molecules.

As used herein, “target mRNA” refers to an RNA molecule that is a targetfor downregulation.

siRNA can be expressed from pol III expression vectors without a changein targeting site, as expression of RNAs from pol III promoters is onlybelieved to be efficient when the first transcribed nucleotide is apurine.

siRNA according to the present technology can be targeted to any stretchof approximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA are given, for example, in Tuschl T. et al., “ThesiRNA User Guide”, revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Laboratory of RNA Molecular Biology, Rockefeller University, New York,USA, and can be found by accessing the website of the RockefellerUniversity and searching with the keyword “siRNA”. Thus, the sensestrand of the present siRNA comprises a nucleotide sequencesubstantially identical to any contiguous stretch of about 19 to about25 nucleotides in the target mRNA.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′-direction) from thestart codon. The target sequence can, however, be located in the 5′- or3′-untranslated regions, or in the region nearby the start codon.

siRNA can be obtained using a number of techniques known to those ofskill in the art. For example, siRNA can be chemically synthesized orrecombinantly produced using methods known in the art, such as theDrosophila in vitro system described in U.S. published application2002/0086356 of Tuschl et al., the entire disclosure of which is hereinincorporated by reference.

Preferably, siRNA is chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. siRNA can be synthesized as two separate, complementary RNAmolecules, or as a single RNA molecule with two complementary regions.

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Such embodiments areincluded according to the present technology when reference is madeherein to the administration of siRNA or the incorporation of siRNA intopharmaceutical compositions. Suitable promoters for expressing siRNA ofthe present technology from a plasmid include, for example, the U6 or H1RNA pol III promoter sequences and the cytomegalovirus promoter.

Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the present technology can also compriseinducible or regulatable promoters for expression of the siRNA in aparticular tissue or in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly. The use of recombinant plasmids to deliversiRNA to cells in vivo is discussed in more detail below. siRNA can beexpressed from a recombinant plasmid either as two separate,complementary RNA molecules, or as a single RNA molecule with twocomplementary regions.

Selection of plasmids suitable for expressing siRNA, methods forinserting nucleic acid sequences for expressing the siRNA into theplasmid, and methods of delivering the recombinant plasmid to the cellsof interest are within the skill in the art.

siRNA can also be expressed from recombinant viral vectorsintracellularly in vivo. The recombinant viral vectors comprisesequences encoding the siRNA and any suitable promoter for expressingthe siRNA sequences. The recombinant viral vectors can also compriseinducible or regulatable promoters for expression of the siRNA in aparticular tissue or in a particular intracellular environment. siRNAcan be expressed from a recombinant viral vector either as two separate,complementary RNA molecules, or as a single RNA molecule with twocomplementary regions.

The term “peptide” comprises oligo- and polypeptides and refers tosubstances comprising two or more, preferably 3 or more, preferably 4 ormore, preferably 6 or more, preferably 8 or more, preferably 10 or more,preferably 13 or more, preferably 16 more, preferably 21 or more and upto preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acidsjoined covalently by peptide bonds. The term “protein” refers to largepeptides, preferably to peptides with more than 100 amino acid residues,but in general the terms “peptides” and “proteins” are synonyms and areused interchangeably herein.

Preferably, the proteins and peptides described according to the presenttechnology have been isolated. The terms “isolated protein” or “isolatedpeptide” mean that the protein or peptide has been separated from itsnatural environment. An isolated protein or peptide may be in anessentially purified state. The term “essentially purified” means thatthe protein or peptide is essentially free of other substances withwhich it is associated in nature or in vivo.

Such proteins and peptides may be used, for example, in producingantibodies and in an immunological or diagnostic assay or astherapeutics. Proteins and peptides described according to the presenttechnology may be isolated from biological samples such as tissue orcell homogenates and may also be expressed recombinantly in amultiplicity of pro- or eukaryotic expression systems.

For the purposes of the present technology, “derivatives” of a proteinor peptide or of an amino acid sequence comprise amino acid insertionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example: (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Some preferredsubstitutions may be made among the following groups: (i) S and T; (ii)P and G; and (iii) A, V, L and I. Given the known genetic code, andrecombinant and synthetic DNA techniques, the skilled scientist readilycan construct DNAs encoding the conservative amino acid variants.

Preferably the degree of similarity, preferably identity between aspecific amino acid sequence described herein and an amino acid sequencewhich is a derivative of said specific amino acid sequence will be atleast 70%, preferably at least 80%, preferably at least 85%, even morepreferably at least 90% or most preferably at least 95%, 96%, 97%, 98%or 99%. The degree of similarity or identity is given preferably for aregion of at least about 20, at least about 40, at least about 60, atleast about 80, at least about 100, at least about 120, at least about140, at least about 160, at least about 200 or 250 amino acids. Inpreferred embodiments, the degree of similarity or identity is given forthe entire length of the reference amino acid sequence.

In one embodiment, a protein or peptide which is a derivative of aspecific protein or peptide or which is a part of a specific protein orpeptide has a relevant function and/or activity of the specific proteinor peptide, i.e. it may have the same activity or immunologicalproperties as the specific protein or peptide.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. The manipulation of DNA sequences for preparingproteins and peptides having substitutions, insertions or deletions, isdescribed in detail in Sambrook et al. (1989), for example.

According to the present technology, “derivatives” of proteins andpeptides also comprise single or multiple substitutions, deletionsand/or additions of any molecules associated with the protein orpeptide, such as carbohydrates, lipids and/or proteins or peptides. Theterm “derivative” also extends to all functional chemical equivalents ofsaid proteins and peptides.

According to the present technology, a part or fragment of atumor-associated antigen preferably has a functional property of theprotein or peptide from which it has been derived. Such functionalproperties comprise the interaction with antibodies, the interactionwith other peptides or proteins, the selective binding of nucleic acidsand an enzymatic activity. A particular property is the ability to forma complex with MHC molecules and, where appropriate, generate an immuneresponse, preferably by stimulating cytotoxic or T helper cells. A partor fragment of a tumor-associated antigen preferably comprises asequence of at least 6, in particular at least 8, at least 10, at least12, at least 15, at least 20, at least 30 or at least 50, consecutiveamino acids of the tumor-associated antigen. A part or fragment of atumor-associated antigen preferably comprises a sequence of up to 8, inparticular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55,consecutive amino acids of the tumor-associated antigen. A part orfragment of a tumor-associated antigen is preferably a part of thetumor-associated antigen which corresponds to the non-transmembraneportion, in particular the extracellular portion of the antigen, or iscomprised thereof.

Preferred parts or fragments of a tumor-associated antigen are inparticular suitable for the stimulation of cytotoxic T-lymphocytes invivo but also for the production of expanded and stimulatedT-lymphocytes for the therapeutic adoptive transfer ex vivo.

A part or a fragment of a nucleic acid coding for a tumor-associatedantigen relates according to the present technology to the part of thenucleic acid, which codes at least for the tumor-associated antigenand/or for a part or a fragment of said tumor-associated antigen, asdefined above. A part or fragment of a nucleic acid coding for atumor-associated antigen is preferably that part of the nucleic acidcorresponding to the open reading frame.

According to the present technology, particular embodiments ought toinvolve providing “dominant negative” proteins or peptides derived fromtumor-associated antigens. A dominant negative protein or peptide is aninactive protein or peptide variant which, by way of interacting withthe cellular machinery, displaces an active protein or peptide from itsinteraction with the cellular machinery or which competes with theactive protein or peptide, thereby reducing the effect of said activeprotein.

Antisera which contain specific antibodies specifically binding to thetarget protein can be prepared by various standard processes; see, forexample, “Monoclonal Antibodies: A Practical Approach” by PhilipShepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A LaboratoryManual” by Ed Harlow, David Lane, ISBN: 0879693142 and “UsingAntibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow,David Lane, Ed Harlow ISBN 0879695447. Thereby it is also possible togenerate affine and specific antibodies which recognize complex membraneproteins in their native form (Azorsa et al., J. Immunol. Methods 229:35-48, 1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989;Gardsvoll, J. Immunol. Methods 234: 107-116, 2000). This is inparticular relevant for the preparation of antibodies which are to beused therapeutically, but also for many diagnostic applications. In thisrespect, it is possible to immunize with the whole protein, withextracellular partial sequences as well as with cells which express thetarget molecule in physiologically folded form.

Monoclonal antibodies are traditionally prepared using the hybridomatechnology. (for technical details see: “Monoclonal Antibodies: APractical Approach” by Philip Shepherd, Christopher Dean ISBN0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, DavidLane ISBN: 0879693142; “Using Antibodies: A Laboratory Manual: PortableProtocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).

It is known that only a small part of an antibody molecule, theparatope, is involved in binding of the antibody to its epitope (cf.Clark, W. R. (1986), The Experimental Foundations of Modern Immunology,Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology,7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fcregions are, for example, effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically removed or which has been produced without thepFc′ region, referred to as F(ab′)₂ fragment, carries both antigenbinding sites of a complete antibody. Similarly, an antibody from whichthe Fc region has been enzymatically removed or which has been producedwithout said Fc region, referred to as Fab fragment, carries one antigenbinding site of an intact antibody molecule. Furthermore, Fab fragmentsconsist of a covalently bound light chain of an antibody and part of theheavy chain of said antibody, referred to as Fd. The Fd fragments arethe main determinants of antibody specificity (a single Fd fragment canbe associated with up to ten different light chains, without alteringthe specificity of the antibody) and Fd fragments, when isolated, retainthe ability to bind to an epitope.

Located within the antigen-binding part of an antibody arecomplementary-determining regions (CDRs) which interact directly withthe antigen epitope and framework regions (FRs) which maintain thetertiary structure of the paratope. Both the Fd fragment of the heavychain and the light chain of IgG immunoglobulins contain four frameworkregions (FR1 to FR4) which are separated in each case by threecomplementary-determining regions (CDR1 to CDR3). The CDRs and, inparticular, the CDR3 regions and, still more particularly, the CDR3region of the heavy chain are responsible to a large extent for antibodyspecificity.

Non-CDR regions of a mammalian antibody are known to be able to bereplaced by similar regions of antibodies with the same or a differentspecificity, with the specificity for the epitope of the originalantibody being retained. This made possible the development of“humanized” antibodies in which nonhuman CDRs are covalently linked tohuman FR and/or Fc/pFc′ regions to produce a functional antibody.

As another example, WO 92/04381 describes the production and use ofhumanized murine RSV antibodies in which at least part of the murine FRregions have been replaced with FR regions of a human origin. Antibodiesof this kind, including fragments of intact antibodies withantigen-binding capability, are often referred to as “chimeric”antibodies.

According to the present technology, the term “antibody” also includesF(ab′)₂, Fab, Fv, and Fd fragments of antibodies, chimeric antibodies,in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or lightchain-CDR3 regions have been replaced with homologous human or nonhumansequences, chimeric F(ab′)₂-fragment antibodies in which the FR and/orCDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, chimeric Fab-fragment antibodiesin which the FR and/or CDR1 and/or CDR2 and/or light chain-CDR3 regionshave been replaced with homologous human or nonhuman sequences, andchimeric Fd-fragment antibodies in which the FR and/or CDR1 and/or CDR2regions have been replaced with homologous human or nonhuman sequences.The term “antibody” also comprises “single-chain” antibodies.

The present technology also comprises proteins and peptides which bindspecifically to tumor-associated antigens. Binding substances of thiskind may be provided, for example, by degenerate peptide libraries whichmay be prepared simply in solution in an immobilized form or asphage-display libraries. It is likewise possible to preparecombinatorial libraries of peptides with one or more amino acids.Libraries of peptoids and nonpeptidic synthetic residues may also beprepared.

Antibodies may also be coupled to specific diagnostic substances fordisplaying cells and tissues expressing tumor-associated antigens. Theymay also be coupled to therapeutically useful substances.

Diagnostic substances or agents include any label that functions to: (i)provide a detectable signal; (ii) interact with a second label to modifythe detectable signal provided by the first or second label, e.g. FRET(Fluorescence Resonance Energy Transfer); (iii) affect mobility, e.g.electrophoretic mobility, by charge, hydrophobicity, shape, or otherphysical parameters, or (iv) provide a capture moiety, e.g., affinity,antibody/antigen, or ionic complexation. Suitable as label arestructures, such as fluorescent labels, luminescent labels, chromophorelabels, radioisotopic labels, isotopic labels, preferably stableisotopic labels, isobaric labels, enzyme labels, particle labels, inparticular metal particle labels, magnetic particle labels, polymerparticle labels, small organic molecules such as biotin, ligands ofreceptors or binding molecules such as cell adhesion proteins orlectins, label-sequences comprising nucleic acids and/or amino acidresidues which can be detected by use of binding agents, etc. Diagnosticsubstances comprise, in a nonlimiting manner, barium sulfate, iocetamicacid, iopanoic acid, calcium ipodate, sodium diatrizoate, megluminediatrizoate, metrizamide, sodium tyropanoate and radio diagnostic,including positron emitters such as fluorine-18 and carbon-11, gammaemitters such as iodine-123, technetium-99m, iodine-131 and indium-111,nuclides for nuclear magnetic resonance, such as fluorine andgadolinium.

According to the present technology, the terms “therapeutically usefulsubstance”, “therapeutic substance” or “therapeutic agent” means anymolecule which may exert a therapeutic effect. According to the presenttechnology, a therapeutically useful substance is preferably selectivelyguided to a cell which expresses one or more tumor-associated antigensand includes anticancer agents, radioactive iodine-labeled compounds,toxins, cytostatic or cytolytic drugs, etc. Anticancer agents comprise,for example, aminoglutethimide, azathioprine, bleomycin sulfate,busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide,cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin,doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine,mercaptopurine, methotrexate, mitotane, procarbazine HCl, thioguanine,vinblastine sulfate and vincristine sulfate. Other anticancer agents aredescribed, for example, in Goodman and Gilman, “The PharmacologicalBasis of Therapeutics”, 8th Edition, 1990, McGraw-Hill, Inc., inparticular Chapter 52 (Antineoplastic Agents (Paul Calabresi and BruceA. Chabner). Toxins may be proteins such as pokeweed antiviral protein,cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheriaexotoxin or Pseudomonas exotoxin. Toxin residues may also be highenergy-emitting radionuclides such as cobalt-60.

The term “major histocompatibility complex” or “MHC” relates to acomplex of genes present in all vertebrates. MHC proteins or moleculesare involved in signaling between lymphocytes and antigen presentingcells in normal immune reactions by binding peptides and presenting themfor recognition by T cell receptors (TCR). MHC molecules bind peptideswithin an intracellular processing compartment and present thesepeptides on the surface of antigen presenting cells for recognition by Tcells. The human MHC region also termed HLA is located on chromosome 6and includes the class I and class II region. In one preferredembodiment of all aspects of the present technology an MHC molecule isan HLA molecule.

“Reduce” or “inhibit” as used herein means the ability to cause anoverall decrease, preferably of 20% or greater, more preferably of 50%or greater, and most preferably of 75% or greater, in the level, e.g. inthe level of protein or mRNA as compared to a reference sample (e.g., asample not treated with siRNA). This reduction or inhibition of RNA orprotein expression can occur through targeted mRNA cleavage ordegradation. Assays for protein expression or nucleic acid expressionare known in the art and include, for example, ELISA, western blotanalysis for protein expression, and northern blotting or RNaseprotection assays for RNA.

The term “patient” means according to the present technology a humanbeing, a nonhuman primate or another animal, in particular a mammal suchas a cow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouseand rat. In a particularly preferred embodiment, the patient is a humanbeing.

According to the present technology the term “increased” or “increasedamount” preferably refers to an increase by at least 10%, in particularat least 20%, at least 50% or at least 100%. The amount of a substanceis also increased in a test sample such as a biological sample comparedto a reference sample if it is detectable in the test sample but absentor not detectable in the reference sample.

According to the present technology, the term “disease” refers to anypathological state in which tumor-associated nucleic acids and/ortumor-associated antigens are expressed or abnormally expressed.“Abnormal expression” means according to the present technology thatexpression is altered, preferably increased, compared to the state in ahealthy individual. An increase in expression refers to an increase byat least 10%, in particular at least 20%, at least 50% or at least 100%.In one embodiment, expression is only found in tissue of a diseasedindividual, while expression in a healthy individual is repressed or isrepressed in a healthy individual except for placenta. One example ofsuch a disease is cancer, wherein the term “cancer” according to thepresent technology comprises leukemias, seminomas, melanomas, teratomas,lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer,kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skincancer, cancer of the brain, cervical cancer, intestinal cancer, livercancer, colon cancer, stomach cancer, intestine cancer, head and neckcancer, gastrointestinal cancer, lymph node cancer, esophagus cancer,colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer,breast cancer, prostate cancer, cancer of the uterus, ovarian cancer andlung cancer and the matastases thereof. Examples thereof are lungcarcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas,renal cell carcinomas, cervical carcinomas, or metastases of the cancertypes or tumors described above. The term cancer according to thepresent technology also comprises cancer metastases.

By “tumor” is meant an abnormal group of cells or tissue that grows by arapid, uncontrolled cellular proliferation and continues to grow afterthe stimuli that initiated the new growth cease. Tumors show partial orcomplete lack of structural organization and functional coordinationwith the normal tissue, and usually form a distinct mass of tissue,which may be either benign or malignant.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the term “metastasis”according to the present technology relates to “distant metastasis”which relates to a metastasis which is remote from the primary tumor andthe regional lymph node system.

According to the present technology, a biological sample may be a tissuesample, including bodily fluids, and/or a cellular sample and may beobtained in the conventional manner such as by tissue biopsy, includingpunch biopsy, and by taking blood, bronchial aspirate, sputum, urine,feces or other body fluids. According to the present technology, theterm “biological sample” also includes fractions of biological samples.Preferably, the term “biological sample” according to the presenttechnology does not include samples derived from placental tissue.

According to the present technology, the term “immunoreactive cell”means a cell which can mature into an immune cell (such as B cell, Thelper cell, or cytolytic T cell) with suitable stimulation.Immunoreactive cells comprise CD34⁺ hematopoietic stem cells, immatureand mature T cells and immature and mature B cells. If production ofcytolytic or T helper cells recognizing a tumor-associated antigen isdesired, the immunoreactive cell is contacted with a cell expressing atumor-associated antigen under conditions which favor production,differentiation and/or selection of cytolytic T cells and of T helpercells. The differentiation of T cell precursors into a cytolytic T cell,when exposed to an antigen, is similar to clonal selection of the immunesystem.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells and cytotoxic T cells which comprisecytolytic T cells.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of antigen-presenting cells such ascancer cells which present one or more tumor-associated antigens. Inthis connection, for example autologous cytotoxic T lymphocytes specificfor a complex of a tumor-associated antigen and an MHC molecule areadministered to a patient having a cellular abnormality. The productionof such cytotoxic T lymphocytes in vitro is known. An example of amethod of differentiating T cells can be found in WO-A-9633265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes,fluorogenic tetramers of MHC class I molecule/peptide complexes are usedfor obtaining specific clones of cytotoxic T lymphocytes (Altman et al.,Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).

The present technology also includes therapeutic methods referred to asadoptive transfer (Greenberg, J. Immunol. 136(5):1917, 1986; Riddel etal., Science 257:238, 1992; Lynch et al., Eur. J. Immunol. 21:1403-1410,1991; Kast et al., Cell 59:603-614, 1989), wherein cells presenting thedesired complex (e.g. dendritic cells) are combined with cytotoxic Tlymphocytes of the patient to be treated, resulting in a propagation ofspecific cytotoxic T lymphocytes. The propagated cytotoxic T lymphocytesare then administered to a patient having a cellular anomalycharacterized by particular abnormal cells presenting the specificcomplex. The cytotoxic T lymphocytes then lyse the abnormal cells,thereby achieving a desired therapeutic effect.

Furthermore, cells presenting the desired complex (e.g. dendritic cells)may be combined with cytotoxic T lymphocytes of healthy individuals oranother species (e.g. mouse) which may result in propagation of specificcytotoxic T lymphocytes with high affinity. The high affinity T cellreceptor of these propagated specific T lymphocytes may be cloned andoptionally humanized to a different extent, and the T cell receptorsthus obtained then transduced via gene transfer, for example usingretroviral vectors, into T cells of patients. Adoptive transfer may thenbe carried out using these genetically altered T lymphocytes(Stanislawski et al., Nat Immunol. 2:962-70, 2001; Kessels et al., NatImmunol. 2:957-61, 2001).

Adoptive transfer is not the only form of therapy which can be appliedaccording to the present technology. Cytotoxic T lymphocytes may also begenerated in vivo in a manner known per se. One method usesnonproliferative cells expressing the complex. The cells used here willbe those which usually express the complex, such as irradiated tumorcells or cells transfected with one or both genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting MHC molecule). Another preferred form is the introduction ofthe tumor-associated antigen in the form of recombinant RNA which may beintroduced into cells by liposomal transfer or by electroporation, forexample. The resulting cells present the complex of interest and arerecognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associatedantigen or a fragment thereof with an adjuvant in order to makeincorporation into antigen-presenting cells in vivo possible. Thetumor-associated antigen or a fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The tumor-associatedantigen is processed to produce a peptide partner for the MHC molecule,while a fragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toMHC molecules. Preference is given to administration forms in which thecomplete antigen is processed in vivo by a dendritic cell, since thismay also produce T helper cell responses which are needed for aneffective immune response (Ossendorp et al., Immunol Lett. 74:75-9,2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, itis possible to administer an effective amount of the tumor-associatedantigen to a patient by intradermal injection, for example. However,injection may also be carried out intranodally into a lymph node (Maloyet al., Proc Natl Acad Sci USA 98:3299-303, 2001).

The pharmaceutical compositions and methods of treatment describedaccording to the present technology may also be used for immunization orvaccination to therapeutically treat or prevent a disease describedherein. According to the present technology, the terms “immunization” or“vaccination” preferably relate to an increase in or activation of animmune response to an antigen. It is possible to use animal models fortesting an immunizing effect on cancer by using a tumor-associatedantigen or a nucleic acid coding therefor. For example, human cancercells may be introduced into a mouse to generate a tumor, and one ormore nucleic acids coding for tumor-associated antigens may beadministered. The effect on the cancer cells (for example reduction intumor size) may be measured as a measure for the effectiveness of animmunization by the nucleic acid.

As part of the composition for an immunization or a vaccination,preferably one or more tumor-associated antigens or stimulatingfragments thereof are administered together with one or more adjuvantsfor inducing an immune response or for increasing an immune response. Anadjuvant is a substance which is incorporated into the antigen oradministered together with the latter and which enhances the immuneresponse. Adjuvants may enhance the immune response by providing anantigen reservoir (extracellularly or in macrophages), activatingmacrophages and/or stimulating particular lymphocytes. Adjuvants areknown and comprise in a nonlimiting way monophosphoryl lipid A (MPL,SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21(SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al.,Mol. Cells 7:178-186, 1997), incomplete Freund's adjuvant, completeFreund's adjuvant, vitamin E, montanide, alum, CpG oligonucleotides (cf.Kreig et al., Nature 374:546-9, 1995) and various water-in-oil emulsionsprepared from biologically degradable oils such as squalene and/ortocopherol. Preferably, the peptides are administered in a mixture withDQS21/MPL. The ratio of DQS21 to MPL is typically about 1:10 to 10:1,preferably about 1:5 to 5:1 and in particular about 1:1. Foradministration to humans, a vaccine formulation typically contains DQS21and MPL in a range from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids such as B7-1 and B7-2 (CD80 and CD86, respectively).

The present technology also provides for administration of nucleicacids, proteins or peptides. Proteins and peptides may be administeredin a manner known per se. In one embodiment, nucleic acids areadministered by ex vivo methods, i.e. by removing cells from a patient,genetic modification of said cells in order to incorporate atumor-associated antigen and reintroduction of the altered cells intothe patient. This generally comprises introducing a functional copy of agene into the cells of a patient in vitro and reintroducing thegenetically altered cells into the patient. The functional copy of thegene is under the functional control of regulatory elements which allowthe gene to be expressed in the genetically altered cells. Transfectionand transduction methods are known to the skilled worker. The presenttechnology also provides for administering nucleic acids in vivo byusing vectors such as viruses and target-controlled liposomes. Ifaccording to the present technology reference is made to theadministration or incorporation into pharmaceutical compositions ofnucleic acids this includes embodiments wherein the nucleic acid ispresent in such vectors.

In a preferred embodiment, a virus or viral vector for administering anucleic acid coding for a tumor-associated antigen is selected from thegroup consisting of adenoviruses, adeno-associated viruses, pox viruses,including vaccinia virus and attenuated pox viruses, Semliki Forestvirus, retroviruses, Sindbis virus and Ty virus-like particles.Particular preference is given to adenoviruses and retroviruses. Theretroviruses are typically replication-deficient (i.e. they areincapable of generating infectious particles).

Methods of introducing nucleic acids into cells in vitro or in vivocomprise transfection of nucleic acid calcium phosphate precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection, and the like. In particular embodiments,preference is given to directing the nucleic acid to particular cells.In such embodiments, a carrier used for administering a nucleic acid toa cell (e.g. a retrovirus or a liposome) may have a bound target controlmolecule. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell may be incorporated into or attached to the nucleicacid carrier. Preferred antibodies comprise antibodies which bindselectively a tumor-associated antigen. If administration of a nucleicacid via liposomes is desired, proteins binding to a surface membraneprotein associated with endocytosis may be incorporated into theliposome formulation in order to make target control and/or uptakepossible. Such proteins comprise capsid proteins or fragments thereofwhich are specific for a particular cell type, antibodies to proteinswhich are internalized, proteins addressing an intracellular site, andthe like.

The therapeutic compositions of the present technology may beadministered in pharmaceutically compatible preparations. Suchpreparations may usually contain pharmaceutically compatibleconcentrations of salts, buffer substances, preservatives, carriers,supplementing immunity-enhancing substances such as adjuvants, e.g. CpGoligonucleotides, cytokines, chemokines, saponin, GM-CSF and/or RNA and,where appropriate, other therapeutically active compounds.

The therapeutically active compounds of the present technology may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitoneally, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lung aerosol. Antisense nucleic acids are preferablyadministered by slow intravenous administration.

The compositions of the present technology are administered in effectiveamounts. An “effective amount” refers to the amount which achieves adesired reaction or a desired effect alone or together with furtherdoses. In the case of treatment of a particular disease or of aparticular condition characterized by expression of one or moretumor-associated antigens, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition. According to the presenttechnology, a diagnosis or treatment of cancer may also include thediagnosis or treatment of cancer metastases which have already formed orwill form. According to the present technology, the term “treatment”comprises therapeutic and prophylactic treatment, i.e. prevention.

An effective amount of a composition of the present technology willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors.

The pharmaceutical compositions of the present technology are preferablysterile and contain an effective amount of the therapeutically activesubstance to generate the desired reaction or the desired effect.

The doses administered of the compositions of the present technology maydepend on various parameters such as the type of administration, thecondition of the patient, the desired period of administration, etc. Inthe case that a reaction in a patient is insufficient with an initialdose, higher doses (or effectively higher doses achieved by a different,more localized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg,preferably from 10 ng to 100 μg, are formulated and administered for atreatment or for generating or increasing an immune response. If theadministration of nucleic acids (DNA and RNA) coding fortumor-associated antigens is desired, doses of from 1 ng to 0.1 mg areformulated and administered.

The pharmaceutical compositions of the present technology are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible compositions. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the present technology.Pharmacologically and pharmaceutically compatible salts of this kindcomprise in a nonlimiting way those prepared from the following acids:hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,salicylic, citric, formic, malonic, succinic acids, and the like.Pharmaceutically compatible salts may also be prepared as alkali metalsalts or alkaline earth metal salts, such as sodium salts, potassiumsalts or calcium salts.

A pharmaceutical composition of the present technology may comprise apharmaceutically compatible carrier. According to the presenttechnology, the term “pharmaceutically compatible carrier” refers to oneor more compatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to humans. The term“carrier” refers to an organic or inorganic component, of a natural orsynthetic nature, in which the active component is combined in order tofacilitate application. The components of the pharmaceutical compositionof the present technology are usually such that no interaction occurswhich substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the present technology may containsuitable buffer substances such as acetic acid in a salt, citric acid ina salt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the present technology may be in the form of capsules,tablets, lozenges, solutions, suspensions, syrups, elixirs or in theform of an emulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present technology is described in detail by the figures andexamples below, which are used only for illustration purposes and arenot meant to be limiting. Owing to the description and the examples,further embodiments which are likewise included in the presenttechnology are accessible to the skilled worker.

EXAMPLES

The techniques and methods mentioned herein are carried out in a mannerknown per se and are described, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. All methods includingthe use of kits and reagents are carried out according to themanufacturers' information unless specifically indicated.

Example 1 Screening for Placenta-Specific Genes Aberrantly Activated inTumors

Tissues and Cell Lines

Tissues were obtained as human surplus materials during routinediagnostic or therapeutic procedures and were stored at −80° C. untiluse. Cell lines were purchased from the American Type Culture Collection(ATCC) and the German Resource Collection of Microorganisms and CellCulture (DSMZ).

RNA Isolation and Microarray Hybridization

Total RNA was isolated using the RNeasy Mini Kit protocol (Qiagen).Quantification of isolated RNA was performed using UV-spectroscopy andthe quality was determined both by A₂₆₀/A₂₈₀ ratio and Agilentbioanalyzer (Agilent Technologies). Five micrograms total RNA were usedfor cDNA synthesis with 5 pmol μl⁻¹ T7-oligo(dT)₂₄ primer and wasperformed at 43° C. for 90 minutes with the “Superscript First-StrandSynthesis-System” for RT-PCR (Invitrogen). Second-strand synthesis wasperformed with complete cDNA. The cDNA solution was incubated at 160° C.for 2 hours followed by an incubation step for 20 min with 6 U T4-DNApolymerase at 16° C. and the reaction was stopped using 10 μl of 0.5 MEDTA. After purification of the double stranded cDNA using the GeneChipSample Cleanup Module (Affymetrix) labeled cRNA was generated from thecDNA sample by an in vitro transcription reaction that was supplementedwith biotin-11-CTP and biotin-16-UTP (Enzo Diagnostics) according to themanufacturer's instructions. The cRNA was quantified by A₂₆₀, and thequality was determined using the labchip bioanalyzer (Agilent). OnlycRNA specimens with a high quality were selected for further analyses.Fragmented cRNA (15 μg) was used to prepare 300 μl hybridizationcocktail (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20) containing0.1 mg ml⁻¹ of herring sperm DNA, and 0.5 mg ml⁻¹ acetylated bovineserum albumin. Control cRNA was used in order to compare hybridizationefficiencies between arrays and to standardize the quantification ofmeasured transcript levels and was included as component of the‘Eukaryotic Hybridization Control kit’ (Affymetrix, Santa Clara, Calif.,USA). The cocktails were heated to 95° C. for 5 minutes, equilibrated at45° C. for 5 minutes, and clarified by centrifugation. The cocktail washybridized to HG U133 Plus 2.0 arrays (Affymetrix) at 45° C. for 16hours. The arrays were washed and stained with a streptavidin-conjugatedfluor using the GeneChip fluidics station protocol EukGE-WS2(Affymetrix) according to the manufacturer's instructions. Arrays werescanned with an argon-ion laser confocal scanner (Hewlett-Packard, SantaClara, Calif.) with detection at 570 nm. Data were extracted usingMicroarray Suite version 5.0 (Affymetrix) and linearly scaled to achievean average intensity of 2,500 per gene. Text files were exported todetermine the intensity of each interrogating oligonucleotide perfectmatch probe cells or mismatch probe cells. In addition, the ratios of5′- and 3′-ends of mRNA were analyzed of six randomly selected specimens(two of each group) using microarray test-chips (Test3 Array) containing24 human housekeeping/maintenance genes (Affymetrix) and RNA degradationwas not observed.

Bioinformatic Analysis

The GeneChip® Operating Software 1.4 (Affymetrix) and ArrayAssistsoftware package 5.2 (Stratagene) were used for statistical analyses.

Results

Screening of samples from the 18 normal tissues shown below in table 1and 30 tumor cell lines of different entities shown below in table 2resulted in the sequences described herein which are expressed inplacenta among the normal tissues investigated and in tumor cell lines.

TABLE 1 Tissues used for microarray expression analysis Tissue NumberPlacenta 2 Testis 2 Mammary gland 2 Thymus 2 Skin 2 Liver 2 Colon 2Esophagus 2 Stomach 2 Lung 2 Kidney 2 Lymph node 2 Skeletal muscle 2Myocard 1 Brain 1 Cerebellum 1 resting PBMCs 2 activ. PBMCs 2

TABLE 2 Cell lines used for microarray expression analysis Cell lineTissue BT-549 Breast cancer MDA-MB-231 metastasizing Breast cancerMDA-MB-231 non-metastasizing Breast cancer MDA-MB-435S Breast cancerMDA-MB-468 Breast cancer SK-BR-3 Breast cancer Caov-3 Ovarian cancerFU-OV Ovarian cancer NIH-OVCAR-3 Ovarian cancer COLO-205 Colorectalcancer HCT-116 Colorectal cancer HCT-116 DKO Colorectal cancer HCT-15Colorectal cancer HT-29 Colorectal cancer LOVO Colorectal cancer SW-480Colorectal cancer CPC-N Lung cancer LOU-NH-91 Lung cancer SHP-77 Lungcancer SK-MES-1 Lung cancer NCI-H-187 Lung cancer NCI-H-209 Lung cancerNCI-H-522 Lung cancer DU-145 Prostate cancer Uncap Prostate cancer PC-3Prostate cancer MEL-JUSO Melanoma Murkowski Melanoma SK-MEL-37 MelanomaHELA Cervical cancer

Example 2 Validation of the Identified Tumor-Associated Markers

1. Examination of RNA Expression

The identified tumor-associated markers are first validated with the aidof RNA which is obtained from various tissues or from tissue-specificcell lines. Since the differential expression pattern of healthy tissuein comparison with tumor tissue is of decisive importance for thesubsequent therapeutic application, the target genes are preferablycharacterized with the aid of these tissue samples.

Total RNA is isolated from native tissue samples or from tumor celllines by standard methods of molecular biology. Said isolation may becarried out, for example, with the aid of the RNeasy Maxi kit (Qiagen,Cat. No. 75162) according to the manufacturer's instructions. Thisisolation method is based on the use of chaotropic reagent guanidiniumisothiocyanate. Alternatively, acidic phenol can be used for isolation(Chomczynski & Sacchi, Anal. Biochem. 162: 156-159, 1987). After thetissue has been worked up by means of guanidinium isothiocyanate, RNA isextracted with acidic phenol, subsequently precipitated with isopropanoland taken up in DEPC-treated water.

2-4 μg of the RNA isolated in this way are subsequently transcribed intocDNA, for example by means of Superscript II (Invitrogen) according tothe manufacturer's protocol. cDNA synthesis is primed with the aid ofrandom hexamers (e.g. Roche Diagnostics) according to standard protocolsof the relevant manufacturer. For quality control, the cDNAs areamplified over 30 cycles, using primers specific for the p53 gene whichis expressed only lowly. Only p53-positive cDNA samples will be used forthe subsequent reaction steps.

The targets are analyzed in detail by carrying out an expressionanalysis by means of PCR or quantitative PCR (qPCR) on the basis of acDNA archive which has been isolated from various normal and tumortissues and from tumor cell lines. For this purpose, 0.5 μl of cDNA ofthe above reaction mixture is amplified by a DNA polymerase (e.g. 1 U ofHotStarTaq DNA polymerase, Qiagen) according to the protocols of theparticular manufacturer (total volume of the reaction mixture: 25-50μl). Aside from said polymerase, the amplification mixture comprises 0.3mM dNTPs, reaction buffer (final concentration 1×, depending on themanufacturer of the DNA polymerase) and in each case 0.3 mMgene-specific “sense” and “antisense” primers.

The specific primers of the target gene are, as far as possible,selected in such a way that they are located in two different exons sothat genomic contaminations do not lead to false-positive results. In anon-quantitative end point PCR, the cDNA is typically incubated at 95°C. for 15 minutes in order to denature the DNA and to activate theHot-Start enzyme. Subsequently the DNA is amplified over 35 cycles (1min at 95° C., 1 min at the primer-specific hybridization temperature(approx. 55-65° C.), 1 min at 72° C. to elongate the amplicons).Subsequently, 10 μl of the PCR mixture are applied to agarose gels andfractionated in the electric field. The DNA is made visible in the gelsby staining with ethidium bromide and the PCR result is documented byway of a photograph.

As an alternative to conventional PCR, expression of a target gene mayalso be analyzed by quantitative real time PCR. Meanwhile variousanalytical systems are available for this analysis, of which the bestknown ones are the ABI PRISM sequence detection system (TaqMan, AppliedBiosystems), the iCycler (Biorad) and the Light cycler (RocheDiagnostics). As described above, a specific PCR mixture is subjected toa run in the real time instruments. By adding a DNA-intercalating dye(e.g. ethidium bromide, CybrGreen), the newly synthesized DNA is madevisible by specific light excitation (according to the dyemanufacturers' information). A multiplicity of points measured duringamplification enables the entire process to be monitored and the nucleicacid concentration of the target gene to be determined quantitatively.The PCR mixture is normalized by measuring a housekeeping gene (e.g. 18SRNA, β-actin). Alternative strategies via fluorescently labeled DNAprobes likewise allow quantitative determination of the target gene of aspecific tissue sample (see TaqMan applications from AppliedBiosystems).

As shown in FIG. 1, placenta was confirmed in RT-PCR analyses as theonly healthy tissue expressing the nucleic acid sequence according toSEQ ID NO:540. No significant expression was found in any other normaltissue. However, high and significant levels of expression were found inbreast cancer.

Quantitative real-time RT-PCR analyses revealed that the nucleic acidsequence according to SEQ ID NO:540 was expressed in significant levelsin the majority of breast cancer samples analyzed; cf. FIG. 2.

2. Cloning

The complete target gene which is required for further characterizationof the tumor-associated marker is cloned according to commonmolecular-biological methods (e.g. in “Current Protocols in MolecularBiology”, John Wiley & Sons Ltd., Wiley InterScience). In order to clonethe target gene or to analyze its sequence, said gene is first amplifiedby a DNA polymerase having a proof reading function (e.g. pfu, RocheDiagnostics). The amplicon is then ligated by standard methods into acloning vector. Positive clones are identified by sequence analysis andsubsequently characterized with the aid of prediction programs and knownalgorithms.

3. Prediction of the Protein

Genes found according to the present technology (in particular thosefrom the RefSeq XM domain) may require cloning of the full-length gene,determination of the open reading frame and deduction and analysis ofthe protein sequence.

In order to clone the full-length sequence, common protocols for therapid amplification of cDNA ends and the screening of cDNA expressionlibraries with gene-specific probes may be used (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.).

After assembling the fragments found in this way, potential open readingframes (ORF) can be predicted using common prediction programs. Sincethe position of the PolyA tail and of polyadenylation motifspredetermines the orientation of the potential gene product, only the 3reading frames of that particular orientation remain out of a possible 6reading frames. The former often yield only one sufficiently large openreading frame which may code for a protein, while the other readingframes have too many stop codons and would not code for any realisticprotein. In the case of alternative open reading frames, identificationof the authentic ORF is assisted by taking into account the Kozakcriteria for optimal transcription initiation and by analyzing thededuced protein sequences which may arise. Said ORF is further verifiedby generating immune sera against proteins deduced from the potentialORFs and analyzing said immune sera for recognition of a real protein intissues and cell lines.

4. Production of Antibodies

The tumor-associated antigens identified according to the presenttechnology are characterized, for example, by using antibodies. Thepresent technology further comprises the diagnostic or therapeutic useof antibodies. Antibodies may recognize proteins in the native and/ordenatured state (Anderson et al., J. Immunol. 143: 1899-1904, 1989;Gardsvoll, J. Immunol. Methods 234: 107-116, 2000; Kayyem et al., Eur.J. Biochem. 208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224:51-60, 1999).

Antisera comprising specific antibodies which specifically bind to thetarget protein may be prepared by various standard methods; cf., forexample, “Monoclonal Antibodies: A Practical Approach” by PhillipShepherd, Christopher Dean ISBN 0-19-963722-9, “Antibodies: A LaboratoryManual” by Ed Harlow, David Lane ISBN: 0879693142 and “Using Antibodies:A Laboratory Manual: Portable Protocol NO” by Edward Harlow, David Lane,Ed Harlow ISBN: 0879695447. It is also possible here to generate affineand specific antibodies which recognize complex membrane proteins intheir native form (Azorsa et al., J. Immunol. Methods 229: 35-48, 1999;Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J.Immunol. Methods. 234: 107-116, 2000). This is especially important inthe preparation of antibodies which are intended to be usedtherapeutically but also for many diagnostic applications. For thispurpose, both the complete protein and extracellular partial sequencesmay be used for immunization.

Immunization and Production of Polyclonal Antibodies

Various immunization protocols are published. A species (e.g. rabbits,mice) is immunized by a first injection of the desired target protein.The immune response of the animal to the immunogen can be enhanced by asecond or third immunization within a defined period of time (approx.2-4 weeks after the previous immunization). Blood is taken from saidanimals and immune sera obtained, again after various defined timeintervals (1st bleeding after 4 weeks, then every 2-3 weeks, up to 5takings). The immune sera taken in this way comprise polyclonalantibodies which may be used to detect and characterize the targetprotein in Western blotting, by flow cytometry, immunofluorescence orimmunohistochemistry.

The animals are usually immunized by any of four well-establishedmethods, with other methods also in existence. The immunization may becarried out using peptides specific for the target protein, using thecomplete protein, or using extracellular partial sequences of a proteinwhich can be identified experimentally or via prediction programs. Sincethe prediction programs do not always work perfectly, it is alsopossible to employ two domains separated from one another by atransmembrane domain. In this case, one of the two domains has to beextracellular, which may then be proved experimentally (see below).Immunization is offered commercially by different service providers.

-   -   (1) In the first case, peptides (length: 8-12 amino acids) are        synthesized by in vitro methods (possibly carried out by a        commercial service), and said peptides are used for        immunization. Normally 3 immunizations are carried out (e.g.        with a concentration of 5-100 μg/immunization).    -   (2) Alternatively, immunization may be carried out using        recombinant proteins. For this purpose, the cloned DNA of the        target gene is cloned into an expression vector and the target        protein is synthesized, for example, cell-free in vitro, in        bacteria (e.g. E. coli), in yeast (e.g. S. pombe), in insect        cells or in mammalian cells, according to the conditions of the        particular manufacturer (e.g. Roche Diagnostics, Invitrogen,        Clontech, Qiagen). It is also possible to synthesize the target        protein with the aid of viral expression systems (e.g.        baculovirus, vacciniavirus, adenovirus). After it has been        synthesized in one of said systems, the target protein is        purified, normally by employing chromatographic methods. In this        context, it is also possible to use for immunization proteins        which have a molecular anchor as an aid for purification (e.g.        His tag, Qiagen; FLAG tag, Roche Diagnostics; GST fusion        proteins). A multiplicity of protocols can be found, for        example, in “Current Protocols in Molecular Biology”, John Wiley        & Sons Ltd., Wiley InterScience. After the target protein has        been purified, an immunization is carried out as described        above.    -   (3) If a cell line is available which synthesizes the desired        protein endogenously, it is also possible to use this cell line        directly for preparing the specific antiserum. In this case,        immunization is carried out by 1-3 injections with in each case        approx. 1−5×10⁷ cells.    -   (4) The immunization may also be carried out by injecting DNA        (DNA immunization). For this purpose, the target gene is first        cloned into an expression vector so that the target sequence is        under the control of a strong eukaryotic promoter (e.g. CMV        promoter). Subsequently, DNA (e.g. 1-10 μg per injection) is        transferred as immunogen using a gene gun into capillary regions        with a strong blood flow in an organism (e.g. mouse, rabbit).        The transferred DNA is taken up by the animal's cells, the        target gene is expressed, and the animal finally develops an        immune response to the target protein (Jung et al., Mol. Cells        12: 41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21:        287-293, 2002).

Production Of Monoclonal Antibodies

Monoclonal antibodies are traditionally produced with the aid of thehybridoma technology (technical details: see “Monoclonal Antibodies: APractical Approach” by Philip Shepherd, Christopher Dean ISBN0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, DavidLane ISBN: 0879693142, “Using Antibodies: A Laboratory Manual PortableProtocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).A new method which is also used is the “SLAM” technology. Here, B cellsare isolated from whole blood and the cells are made monoclonal.Subsequently the supernatant of the isolated B cell is analyzed for itsantibody specificity. In contrast to the hybridoma technology, thevariable region of the antibody gene is then amplified by single-cellPCR and cloned into a suitable vector. In this manner production ofmonoclonal antibodies is accelerated (de Wildt et al., J. Immunol.Methods 207:61-67, 1997).

5. Validation of the Targets by Protein-Chemical Methods UsingAntibodies

The antibodies which can be produced as described above can be used tofurther analyze the target protein as follows:

Specificity Of the Antibody

Assays based on cell culture with subsequent Western blotting are mostsuitable for demonstrating the fact that an antibody binds specificallyonly to the desired target protein (various variations are described,for example, in “Current Protocols in Protein Chemistry”, John Wiley &Sons Ltd., Wiley InterScience). For the demonstration, cells aretransfected with a cDNA for the target protein, which is under thecontrol of a strong eukaryotic promoter (e.g. cytomegalovirus promoter;CMV). A wide variety of methods (e.g. electroporation, liposome-basedtransfection, calcium phosphate precipitation) are well established fortransfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol.Biol. 75: 441-7, 1997). As an alternative, it is also possible to usecell lines which express the target gene endogenously (detection viatarget gene-specific RT-PCR). As a control, in the ideal case,homologous genes are cotransfected in the experiment, in order to beable to demonstrate in the following Western blot the specificity of theanalyzed antibody.

In the subsequent Western blotting, cells from cell culture or tissuesamples which might contain the target protein are lysed in a 1%strength SDS solution, and the proteins are denatured in the process.The lysates are fractionated according to size by electrophoresis on8-15% strength denaturing polyacrylamide gels (contain 1% SDS) (SDSpolyacrylamide gel electrophoresis, SDS-PAGE). The proteins are thentransferred by one of a plurality of blotting methods (e.g. semi-dryelectroblot; Biorad) to a specific membrane (e.g. nitrocellulose,Schleicher & Schüll). The desired protein can be visualized on thismembrane. For this purpose, the membrane is first incubated with theantibody which recognizes the target protein (dilution approx.1:20-1:200, depending on the specificity of said antibody), for 60minutes. After a washing step, the membrane is incubated with a secondantibody which is coupled to a marker (e.g. enzymes such as peroxidaseor alkaline phosphatase) and which recognizes the first antibody. It isthen possible to make the target protein visible on the membrane in acolor or chemi-luminescent reaction (e.g. ECL, Amersham Bioscience). Anantibody with a high specificity for the target protein should in theideal case only recognize the desired protein itself.

Localization of the Target Protein

Various methods are used to confirm the membrane localization,identified in the in silico approach, of the target protein. Animportant and well-established method using the antibodies describedabove is immunofluorescence (IF). For this purpose, cells of establishedcell lines which either synthesize the target protein (detection of theRNA by RT-PCR or of the protein by Western blotting) or else have beentransfected with plasmid DNA are utilized. A wide variety of methods(e.g. electroporation, liposome-based transfection, calcium phosphateprecipitation) are well established for transfection of cell lines withDNA (e.g. Lemoine et al., Methods Mol. Biol. 75: 441-7, 1997). Theplasmid transfected into the cells, in immunofluorescence, may encodethe unmodified protein or else couple different amino acid markers tothe target protein. The principle markers are, for example, thefluorescent green fluorescent protein (GFP) in various differentiallyfluorescent forms, short peptide sequences of 6-12 amino acids for whichhigh-affinity and specific antibodies are available, or the short aminoacid sequence Cys-Cys-X-X-Cys-Cys which can bind via its cysteinesspecific fluorescent substances (Invitrogen). Cells which synthesize thetarget protein are fixed, for example, with paraformaldehyde ormethanol. The cells may then, if required, be permeabilized byincubation with detergents (e.g. 0.2% Triton X-100). The cells are thenincubated with a primary antibody which is directed against the targetprotein or against one of the coupled markers. After a washing step, themixture is incubated with a second antibody coupled to a fluorescentmarker (e.g. fluorescein, Texas Red, Dako), which binds to the firstantibody. The cells labeled in this way are then overlaid with glyceroland analyzed with the aid of a fluorescence microscope according to themanufacturer's information. Specific fluorescence emissions are achievedin this case by specific excitation depending on the substancesemployed. The analysis usually permits reliable localization of thetarget protein, the antibody quality and the target protein beingconfirmed in double stainings with, in addition to the target protein,also the coupled amino acid markers or other marker proteins whoselocalization has already been described in the literature being stained.GFP and its derivatives represent a special case, being excitabledirectly and themselves fluorescing. The membrane permeability which maybe controlled through the use of detergents, in immunofluorescence,allows demonstration of whether an immunogenic epitope is located insideor outside the cell. The prediction of the selected proteins can thus besupported experimentally. An alternative possibility is to detectextracellular domains by means of flow cytometry. For this purpose,cells are fixed under non-permeabilizing conditions (e.g. with PBS/Naazide/2% FCS/5 mM EDTA) and analyzed in a flow cytometer in accordancewith the manufacturer's instructions. Only extracellular epitopes can berecognized by the antibody to be analyzed in this method. A differencefrom immunofluorescence is that it is possible to distinguish betweendead and living cells by using, for example, propidium iodide or trypanblue, and thus avoid false-positive results.

Another important detection is by immunohistochemistry (IHC) on specifictissue samples. The aim of this method is to identify the localizationof a protein in a functionally intact tissue aggregate. IHC servesspecifically for (1) being able to estimate the amount of target proteinin tumor and normal tissues, (2) analyzing how many cells in tumor andhealthy tissues synthesize the target gene, and (3) defining the celltype in a tissue (tumor, healthy cells) in which the target protein isdetectable. Alternatively, the amounts of protein of a target gene maybe quantified by tissue immunofluorescence using a digital camera andsuitable software (e.g. Tillvision, Till-photonics, Germany). Thetechnology has frequently been published, and details of staining andmicroscopy can therefore be found, for example, in “DiagnosticImmunohistochemistry” by David J., MD Dabbs ISBN: 0443065667 or in“Microscopy, Immunohistochemistry, and Antigen Retrieval Methods ForLight and Electron Microscopy” ISBN: 0306467704. It should be notedthat, owing to the properties of antibodies, different protocols have tobe used (an example is described below) in order to obtain a meaningfulresult.

Normally, histologically defined tumor tissues and, as reference,comparable healthy tissues are employed in IHC. It is also possible touse as positive and negative controls cell lines in which the presenceof the target gene is known through RT-PCR analyses. A backgroundcontrol must always be included.

Formalin-fixed (another fixation method, for example with methanol, isalso possible) and paraffin-embedded tissue pieces with a thickness of 4μm are applied to a glass support and deparaffinated with xylene, forexample. The samples are washed with TBS-T and blocked in serum. This isfollowed by incubation with the first antibody (dilution: 1:2 to 1:2000)for 1-18 hours, with affinity-purified antibodies normally being used. Awashing step is followed by incubation with a second antibody which iscoupled to an alkaline phosphatase (alternative: for example peroxidase)and directed against the first antibody, for approx. 30-60 minutes. Thisis followed by a color reaction using alkaline phosphatase (cf., forexample, Shi et al., J. Histochem. Cytochem. 39: 741-748, 1991; Shin etal., Lab. Invest. 64: 693-702, 1991). To demonstrate antibodyspecificity, the reaction can be blocked by previous addition of theimmunogen.

Analysis of Protein Modifications

Secondary protein modifications such as, for example, N- orO-glycosylations or myristilations may impair or even completely preventthe accessibility of immunogenic epitopes and thus call into questionthe efficacy of antibody therapies. Moreover, it has frequently beendemonstrated that the type and amount of secondary modifications differin normal and tumor tissues (e.g. Durand & Seta, 2000; Clin. Chem. 46:795-805; Hakomori, 1996; Cancer Res. 56: 5309-18). The analysis of thesemodifications is therefore essential to the therapeutic success of anantibody. Potential binding sites can be predicted by specificalgorithms.

Analysis of protein modifications usually takes place by Westernblotting (see above). Glycosylations which usually have a size ofseveral kDa, especially lead to a larger total mass of the targetprotein, which can be fractionated in SDS-PAGE. To detect specific O-and N-glycosidic bonds, protein lysates are incubated prior todenaturation by SDS with O- or N-glycosylases (in accordance with theirrespective manufacturer's instructions, e.g. PNgase, endoglycosidase F,endoglycosidase H, Roche Diagnostics). This is followed by Westernblotting as described above. Thus, if there is a reduction in the sizeof a target protein after incubation with a glycosidase, it is possibleto detect a specific glycosylation and, in this way, also analyze thetumor specificity of a modification.

Functional Analysis of the Target Gene

The function of the target molecule may be crucial for its therapeuticusefulness, so that functional analyses are an important component inthe characterization of therapeutically utilizable molecules. Thefunctional analysis may take place either in cells in cell cultureexperiments or else in vivo with the aid of animal models. This involveseither switching off the gene of the target molecule by mutation(knockout) or inserting the target sequence into the cell or theorganism (knockin). Thus it is possible to analyze functionalmodifications in a cellular context firstly by way of the loss offunction of the gene to be analyzed (loss of function). In the secondcase, modifications caused by addition of the analyzed gene can beanalyzed (gain of function).

a. Functional Analysis in Cells

Transfection. In order to analyze the gain of function, the gene of thetarget molecule must be transferred into the cell. For this purpose,cells which allow synthesis of the target molecule are transfected witha DNA. Normally, the gene of the target molecule here is under thecontrol of a strong eukaryotic promoter (e.g. cytomegalovirus promoter;CMV). A wide variety of methods (e.g. electroporation, liposome-basedtransfection, calcium phosphate precipitation) are well established fortransfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol.Biol. 75: 441-7, 1997). The gene may be synthesized either transiently,without genomic integration, or else stably, with genomic integrationafter selection with neomycin, for example.

RNA interference (siRNA). An inhibition of expression of the targetgene, which may induce a complete loss of function of the targetmolecule in cells, may be generated by the RNA interference (siRNA)technology in cells (Hannon, G J. 2002. RNA interference. Nature 418:244-51; Czauderna et al. 2003. Nucl. Acid Res. 31: 670-82). For thispurpose, cells are transfected with short, double-stranded RNA moleculesof approx. 20-25 nucleotides in length, which are specific for thetarget molecule. An enzymic process then results in degradation of thespecific RNA of the target gene and thus in reduced expression of thetarget protein and consequently enables the target gene to befunctionally analyzed.

Cell lines which have been modified by means of transfection or siRNAmay subsequently be analyzed in different ways. The most common examplesare listed below.

1. Proliferation and Cell Cycle Behavior

A multiplicity of methods for analyzing cell proliferation areestablished and are commercially supplied by various companies (e.g.Roche Diagnostics, Invitrogen; details of the assay methods aredescribed in the numerous application protocols). The number of cells incell culture experiments can be determined by simple counting or bycolorimetric assays which measure the metabolic activity of the cells(e.g. wst-1, Roche Diagnostics). Metabolic assay methods measure thenumber of cells in an experiment indirectly via enzymic markers. Cellproliferation may be measured directly by analyzing the rate of DNAsynthesis, for example by adding bromodeoxyuridine (BrdU), with theintegrated BrdU being detected colorimetrically via specific antibodies.

2. Apoptosis and Cytotoxicity

A large number of assay systems for detecting cellular apoptosis andcytotoxicity are available. A decisive characteristic is the specific,enzyme-dependent fragmentation of genomic DNA, which is irreversible andin any case results in death of the cell. Methods for detecting thesespecific DNA fragments are commercially obtainable. An additional methodavailable is the TUNEL assay which can detect DNA single-strand breaksalso in tissue sections. Cytotoxicity is mainly detected via an alteredcell permeability which serves as marker of the vitality state of cells.This involves on the one hand the analysis of markers which cantypically be found intracellularly in the cell culture supernatant. Onthe other hand, it is also possible to analyze the absorbability of dyemarkers which are not absorbed by intact cells. The best-known examplesof dye markers are Trypan blue and propidium iodide, a commonintracellular marker is lactate dehydrogenase which can be detectedenzymatically in the supernatant. Different assay systems of variouscommercial suppliers (e.g. Roche Diagnostics, Invitrogen) are available.

3. Migration Assay

The ability of cells to migrate is analyzed in a specific migrationassay, preferably with the aid of a Boyden chamber (Corning Costar)(Cinamon G., Alon R. J. Immunol. Methods. 2003 February; 273(1-2):53-62;Stockton et al. 2001. Mol. Biol. Cell. 12: 1937-56). For this purpose,cells are cultured on a filter with a specific pore size. Cells whichcan migrate are capable of migrating through this filter into anotherculture vessel below. Subsequent microscopic analysis then permitsdetermination of a possibly altered migration behavior induced by thegain of function or loss of function of the target molecule.

b. Functional Analysis in Animal Models

A possible alternative of cell culture experiments for the analysis oftarget gene function are complicated in vivo experiments in animalmodels. Compared to the cell-based methods, these models have theadvantage of being able to detect faulty developments or diseases whichare detectable only in the context of the whole organism. A multiplicityof models for human disorders are available by now (Abate-Shen & Shen.2002. Trends in Genetics S1-5; Matsusue et al. 2003. J. Clin. Invest.111:737-47). Various animal models such as, for example, yeast,nematodes or zebra fish have since been characterized intensively.However, models which are preferred over other species are mammaliananimal models such as, for example, mice (Mus musculus) because theyoffer the best possibility of reproducing the biological processes in ahuman context. For mice, on the one hand transgenic methods whichintegrate new genes into the mouse genome have been established inrecent years (gain of function; Jegstrup I. et al. 2003. Lab Anim. 2003January; 37(1):1-9). On the other hand, other methodical approachesswitch off genes in the mouse genome and thus induce a loss of functionof a desired gene (knockout models, loss of function; Zambrowicz B P &Sands A T. 2003. Nat. Rev. Drug Discov. 2003 January; 2(1):38-51; NiwaH.2001. Cell Struct. Funct. 2001 June; 26(3):137-48); technical detailshave been published in large numbers.

After the mouse models have been generated, alterations induced by thetransgene or by the loss of function of a gene can be analyzed in thecontext of the whole organism (Balling R, 2001. Ann. Rev. Genomics Hum.Genet. 2:463-92). Thus it is possible to carry out, for example,behavior tests as well as to biochemically study established bloodparameters. Histological analyses, immunohistochemistry or electronmicroscopy enable alterations to be characterized at the cellular level.The specific expression pattern of a gene can be detected by in-situhybridization (Peters T. et al. 2003. Hum. Mol. Genet 12:2109-20).

Example 3 Detailed Analysis of the Identified Tumor-Associated Markers

RNA-Isolation, RT-PCR and Real-Time RT-PCR

RNA extraction, first-strand cDNA synthesis, RT-PCR and real-time RT-PCRwas performed as previously described (Koslowski, M. et al., Cancer Res.62, 6750-6755 (2002), Koslowski, M. et al., Cancer Res. 64, 5988-5993(2004)). Real-time quantitative expression analysis was performed in a40 cycle RT-PCR. After normalization to HPRT (sense 5′-TGA CAC TGG CAAAAC AAT GCA-3′; antisense 5′-GGT CCT TTT CAC CAG CAA GCT-3′, 62° C.annealing) gene-specific transcripts in tumor samples were quantifiedrelative to normal tissues using ΔΔCT calculation.

siRNA Duplexes

The SEQ ID NO:540 siRNA duplexes (Qiagen, Hilden, Germany) were directedagainst target sequences 5′-NNC CAC AGA AGG UAC CAG UUA-3′ (siRNA #1;sense (5′-CCA CAG AAG GUA CCA GUU AUU-3′), antisense (5′-UAA CUG GUA CCUUCU GUG GUU-3′) and 5′-NNC AGC AAG ACU CCC UCU AAA-3′ (siRNA #2; sense(5′-CAG CAA GAC UCC CUC UAA AUU-3′), antisense (5′-UUU AGA GGG AGU CUUGCU GUU-3′) of the SEQ ID NO:540 mRNA sequence.

Cell Proliferation Analysis

24 h after transfection with siRNA duplexes 1×10⁴ cells were culturedfor 48 h in medium supplemented with 10% FCS. Proliferation was analyzedby measuring the incorporation of BrdU into newly synthesized DNAstrands using the DELFIA cell proliferation Kit (Perkin Elmer, Boston,Mass.) according to the manufacturer's instructions on a Wallac Victor²multi-label counter (Perkin Elmer, Boston, Mass.).

FIG. 3 shows the quantification of SEQ ID NO:540 mRNA expression inMCF-7 breast cancer cells by real-time RT-PCR 24 h after transfectionwith siRNA oligos. Compared to non-transfected cells and cellstransfected with non-silencing (ns) siRNA both SEQ ID NO:540-specificsiRNAs (siRNA #1 (SEQ ID NO:630, 631), siRNA #2 (SEQ ID NO:632, 633))induce robust silencing of SEQ ID NO:540 expression.

FIG. 4 shows that silencing of SEQ ID NO:540 expression by transfectionwith siRNA oligos results in impaired proliferation of MCF-7 breastcancer cells. Proliferation was quantified 96 h after transfection withsiRNAs by measuring incorporation of BrdU in newly synthesized DNAstrands. These results show that SEQ ID NO:540 is a positive factor forthe proliferation of breast cancer cells.

The nucleotide sequence according to SEQ ID NO:541 was deduced from SEQID NO:65 and codes for a 177 aa protein (SEQ ID NO:542) of unknownfunction. Expression of SEQ ID NO:541 in normal and cancerous tissueswas quantified by real-time RT-PCR using sequence-specific oligos (SEQID NO:543, 544); see FIG. 5. In normal tissues SEQ ID NO:541 is highlyexpressed in placenta and shows only weak expression in thymus. SEQ IDNO:541 is overexpressed in lung cancer. Based on these expressionresults, SEQ ID NO:541 and its expression products qualify as molecularmarkers and/or target candidates for targeted therapies, in particularfor this particular tumor type.

The nucleotide sequence according to SEQ ID NO:545 was deduced from SEQID NO:249 and codes for a member of the solute carrier (SLC) group ofmembrane proteins (SEQ ID NO:546). As is typical of integral membraneproteins, SLCs contain a number of hydrophobic transmembrane alphahelices connected to each other by hydrophilic intra- or extra-cellularloops. Depending on the SLC, these transporters are functional as eithermonomers or obligate homo- or hetero-oligomers. The protein encoded bySEQ ID NO:545 is a cell surface protein. Expression of SEQ ID NO:545 innormal and cancerous tissues was quantified by real-time RT-PCR usingsequence-specific oligos (SEQ ID NO:547, 548); see FIG. 6. Compared tonormal tissues, SEQ ID NO:545 is overexpressed in malignant melanomas.Based on these expression results, SEQ ID NO:545 and its expressionproducts qualify as molecular markers and/or target candidates fortargeted therapies, in particular for this particular tumor type.

The nucleotide sequence according to SEQ ID NO:549 was deduced from SEQID NO:4 and codes for a 763 aa protein (SEQ ID NO:550) of unknownfunction. The protein harbors two potential transmembrane domains and atypical fibronectin type III domain. Fibronectin is ahigh-molecular-weight extracellular matrix glycoprotein that binds tomembrane spanning receptor proteins (integrins). In addition tointegrins, they also bind extracellular matrix components such ascollagen, fibrin and heparan sulfate. The protein encoded by SEQ IDNO:549 might represent a hitherto unknown new fibronection-like protein.Expression of SEQ ID NO:549 in normal and cancerous tissues wasquantified by real-time RT-PCR using sequence-specific oligos (SEQ IDNO:551, 552); see FIG. 7. Compared to normal tissues, SEQ ID NO:549 isoverexpressed in ovarian cancer. Based on these expression results, SEQID NO:549 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular of thisparticular tumor type.

The nucleotide sequence according to SEQ ID NO:553 was deduced from SEQID NO:156 and codes for a 496 aa protein (SEQ ID NO:554) of unknownfunction. The protein harbors a potential transmembrane protein.Expression of SEQ ID NO:553 in normal and cancerous tissues wasquantified by real-time RT-PCR using sequence-specific oligos (SEQ IDNO:555, 556); see FIG. 8. In normal tissues SEQ ID NO:553 is highlyexpressed in placenta. Compared to other normal tissues, SEQ ID NO:553is overexpressed in colon cancer and ovarian cancer. Based on theseexpression results, SEQ ID NO:553 and its expression products qualify asmolecular markers and/or target candidates for targeted therapies, inparticular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:557 was deduced from SEQID NO:273. SEQ ID NO:557 represents a partial cDNA with no apparent openreading frame. Expression of SEQ ID NO:557 in normal and canceroustissues was quantified by real-time RT-PCR using sequence-specificoligos (SEQ ID NO:558, 559); see FIG. 9. In normal tissues highexpression of SEQ ID NO:557 is detectable in breast. Compared to normaltissues, SEQ ID NO:557 is overexpressed in breast cancer. Based on theseexpression results, SEQ ID NO:557 and its expression products qualify asmolecular markers and/or target candidates for targeted therapies, inparticular for this particular tumor type.

The nucleotide sequence according to SEQ ID NO:560 was deduced from SEQID NO:135. SEQ ID NO:560 has no apparent open reading frame. Expressionof SEQ ID NO:560 in normal and cancerous tissues was quantified byreal-time RT-PCR using sequence-specific oligos (SEQ ID NO:561, 562);see FIG. 10. In normal tissues expression of SEQ ID NO:560 is detectablein duodenum and colon. Compared to normal tissues, SEQ ID NO:560 isoverexpressed in colon cancer and ovarian cancer. Based on theseexpression results, SEQ ID NO:560 and its expression products qualify asmolecular markers and/or target candidates for targeted therapies, inparticular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:563 was deduced from SEQID NO:177. SEQ ID NO:563 has no apparent open reading frame. Expressionof SEQ ID NO:563 in normal and cancerous tissues was quantified byreal-time RT-PCR using sequence-specific oligos (SEQ ID NO:564, 565);see FIG. 11. SEQ ID NO:563 is highly expressed in placenta. Compared tonormal tissues, SEQ ID NO:563 is overexpressed in breast cancer, coloncancer, ovarian cancer, lung cancer and melanoma. Based on theseexpression results, SEQ ID NO:563 and its expression products qualify asmolecular markers and/or target candidates for targeted therapies, inparticular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:566 was deduced from SEQID NO:149 and codes for a 155 aa protein (SEQ ID NO:567) of unknownfunction. The protein sequence is partially homologous to members of thetumor necrosis factor receptor superfamily and harbors a potentialtransmembrane domain. The protein encoded by SEQ ID NO:566 mightrepresent a new member of the tumor necrosis factor receptorsuperfamily. Expression of SEQ ID NO:566 in normal and cancerous tissueswas quantified by real-time RT-PCR using sequence-specific oligos (SEQID NO:568, 569); see FIG. 12. Compared to normal tissues, SEQ ID NO:566is overexpressed in gastric cancer, breast cancer, colon cancer, ovariancancer, lung cancer and melanoma. Based on these expression results, SEQID NO:566 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular for theseparticular tumor types.

The nucleotide sequence according to SEQ ID NO:570 was deduced from SEQID NO:53 and codes for a member of the kernel lipocain superfamily (SEQID NO:571). These secreted glycoproteins have distinct and essentialroles in regulating an uterine environment suitable for pregnancy and inthe timing and occurrence of the appropriate sequence of events in thefertilization process. Expression of SEQ ID NO:570 in normal andcancerous tissues was quantified by real-time RT-PCR usingsequence-specific oligos (SEQ ID NO:572, 573); see FIG. 13. SEQ IDNO:570 is highly expressed in placenta. Compared to other normaltissues, SEQ ID NO:570 is overexpressed in ovarian cancer, lung cancerand melanoma. Based on these expression results, SEQ ID NO:570 and itsexpression products qualify as molecular markers and/or targetcandidates for targeted therapies, in particular for these particulartumor types.

The nucleotide sequence according to SEQ ID NO:574 has no apparent openreading frame. Expression of SEQ ID NO:574 in normal and canceroustissues was quantified by real-time RT-PCR using sequence-specificoligos (SEQ ID NO:575, 576); see FIG. 14. SEQ ID NO:574 is highlyexpressed in placenta. Compared to other normal tissues, SEQ ID NO:574is overexpressed in lung cancer and melanoma. Based on these expressionresults, SEQ ID NO:574 and its expression products qualify as molecularmarkers and/or target candidates for targeted therapies, in particularfor these particular tumor types.

The nucleotide sequence according to SEQ ID NO:577 was deduced from SEQID NO:20. SEQ ID NO:577 represents a partial cDNA with no apparent openreading frame. Expression of SEQ ID NO:577 in normal and canceroustissues was quantified by real-time RT-PCR using sequence-specificoligos (SEQ ID NO:578, 579); see FIG. 15. SEQ ID NO:577 is highlyexpressed in placenta. Compared to other normal tissues, SEQ ID NO:577is overexpressed in gastric cancer, breast cancer and lung cancer. Basedon these expression results, SEQ ID NO:577 and its expression productsqualify as molecular markers and/or target candidates for targetedtherapies, in particular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:580 was deduced from SEQID NO:32. SEQ ID NO:580 represents a partial cDNA with no apparent openreading frame. Expression of SEQ ID NO:580 in normal and canceroustissues was quantified by real-time RT-PCR using sequence-specificoligos (SEQ ID NO:581, 582); see FIG. 16. SEQ ID NO:580 is highlyexpressed in placenta. Compared to other normal tissues, SEQ ID NO:580is overexpressed in ovarian cancer and lung cancer. Based on theseexpression results, SEQ ID NO:580 and its expression products qualify asmolecular markers and/or target candidates for targeted therapies, inparticular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:583 was deduced from SEQID NO:257 and codes for a member of the homeobox class of transcriptionfactors (SEQ ID NO:584). Expression of these proteins is spatially andtemporally regulated during embryonic development. Expression of SEQ IDNO:583 in normal and cancerous tissues was quantified by real-timeRT-PCR using sequence-specific oligos (SEQ ID NO:585, 586); see FIG. 17.SEQ ID NO:583 is highly expressed in placenta and prostate. Compared toother normal tissues, SEQ ID NO:583 is overexpressed in colon cancer,ovarian cancer and lung cancer. Based on these expression results, SEQID NO:583 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular for theseparticular tumor types.

The nucleotide sequence according to SEQ ID NO:587 was deduced from SEQID NO:148 and codes for a member of the IGF-II mRNA-binding protein(IMP) family (SEQ ID NO:588). It functions by binding to the 5′ UTR ofthe insulin-like growth factor 2 (IGF2) mRNA and regulating IGF2translation. Expression of SEQ ID NO:587 in normal and cancerous tissueswas quantified by real-time RT-PCR using sequence-specific oligos (SEQID NO:589, 590); see FIG. 18. Compared to normal tissues, SEQ ID NO:587is overexpressed in lung cancer. Based on these expression results, SEQID NO:587 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular for thisparticular tumor type.

The nucleotide sequence according to SEQ ID NO:591 was deduced from SEQID NO:194 and codes for a 372 aa protein (SEQ ID NO:592) of unknownfunction. Expression of SEQ ID NO:591 in normal and cancerous tissueswas quantified by real-time RT-PCR using sequence-specific oligos (SEQID NO:593, 594); see FIG. 19. SEQ ID NO:591 is highly expressed intestis. Compared to other normal tissues, SEQ ID NO:591 is overexpressedin breast cancer, colon cancer, ovarian cancer, lung cancer andmelanoma. Based on these expression results, SEQ ID NO:591 and itsexpression products qualify as molecular markers and/or targetcandidates for targeted therapies, in particular for these particulartumor types.

The nucleotide sequence according to SEQ ID NO:595 was deduced from SEQID NO:191 and codes for a 357 aa protein (SEQ ID NO:596) of unknownfunction. Expression of SEQ ID NO:595 in normal and cancerous tissueswas quantified by real-time RT-PCR using sequence-specific oligos (SEQID NO:597, 598); see FIG. 20. SEQ ID NO:595 is highly expressed intestis. Compared to other normal tissues, SEQ ID NO:595 is overexpressedin gastric cancer, colon cancer, ovarian cancer, lung cancer andmelanoma. Based on these expression results, SEQ ID NO:595 and itsexpression products qualify as molecular markers and/or targetcandidates for targeted therapies, in particular for these particulartumor types.

The nucleotide sequence according to SEQ ID NO:599 was deduced from SEQID NO:18 and has no apparent open reading frame. Expression of SEQ IDNO:599 in normal and cancerous tissues was quantified by real-timeRT-PCR using sequence-specific oligos (SEQ ID NO:600, 601); see FIG. 21.SEQ ID NO:599 is highly expressed in placenta. Compared to other normaltissues, SEQ ID NO:599 is overexpressed in gastric cancer, breastcancer, lung cancer and melanoma. Based on these expression results, SEQID NO:599 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular for theseparticular tumor types.

The nucleotide sequence according to SEQ ID NO:602 was deduced from SEQID NO:133 and codes for a member of the von Willebrand factor domainsuperfamily of extracellular matrix proteins (SEQ ID NO:603). Expressionof SEQ ID NO:602 in normal and cancerous tissues was quantified byreal-time RT-PCR using sequence-specific oligos (SEQ ID NO:604, 605);see FIG. 22. Compared to normal tissues, SEQ ID NO:602 is overexpressedin ovarian cancer and lung cancer. Based on these expression results,SEQ ID NO:602 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular for theseparticular tumor types.

The nucleotide sequence according to SEQ ID NO:606 was deduced from SEQID NO:128 and codes for a member of the Borg family of CDC42 effectorproteins (SEQ ID NO:607). Borg family proteins contain a CRIB (Cdc42/Racinteractive-binding) domain. They bind to, and negatively regulate thefunction of CDC42. CDC42, a small Rho GTPase, regulates the formation ofF-actin-containing structures through its interaction with thedownstream effector proteins. Expression of SEQ ID NO:606 in normal andcancerous tissues was quantified by real-time RT-PCR usingsequence-specific oligos (SEQ ID NO:608, 609); see FIG. 23. Compared tonormal tissues, SEQ ID NO:606 is overexpressed in gastric cancer, coloncancer and lung cancer. Based on these expression results, SEQ ID NO:606and its expression products qualify as molecular markers and/or targetcandidates for targeted therapies, in particular for these particulartumor types.

The nucleotide sequence according to SEQ ID NO:610 was deduced from SEQID NO:118 and has no apparent open reading frame. Expression of SEQ IDNO:610 in normal and cancerous tissues was quantified by real-timeRT-PCR using sequence-specific oligos (SEQ ID NO:611, 612); see FIG. 24.Compared to normal tissues, SEQ ID NO:610 is overexpressed in gastriccancer, breast cancer and lung cancer. Based on these expressionresults, SEQ ID NO:610 and its expression products qualify as molecularmarkers and/or target candidates for targeted therapies, in particularfor these particular tumor types.

The nucleotide sequence according to SEQ ID NO:613 was deduced from SEQID NO:116 and codes for a 76 aa protein (SEQ ID NO:614) of unknownfunction. Expression of SEQ ID NO:613 in normal and cancerous tissueswas quantified by real-time RT-PCR using sequence-specific oligos (SEQID NO:615, 616); see FIG. 25. SEQ ID NO:613 is highly expressed inplacenta. Compared to other normal tissues, SEQ ID NO:613 isoverexpressed in breast cancer, lung cancer and melanoma. Based on theseexpression results, SEQ ID NO:613 and its expression products qualify asmolecular markers and/or target candidates for targeted therapies, inparticular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:617 was deduced from SEQID NO:267. SEQ ID NO:617 represents a partial cDNA with no apparent openreading frame. Expression of SEQ ID NO:617 in normal and canceroustissues was quantified by real-time RT-PCR using sequence-specificoligos (SEQ ID NO:618, 619); see FIG. 26. SEQ ID NO:617 is highlyexpressed in placenta and endometrium. Compared to other normal tissues,SEQ ID NO:617 is overexpressed in lung cancer and melanoma. Based onthese expression results, SEQ ID NO:617 and its expression productsqualify as molecular markers and/or target candidates for targetedtherapies, in particular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:620 was deduced from SEQID NO:182 and codes for a 829 aa protein (SEQ ID NO:621) harboringmultiple putative transmembrane domains and a patched family domain. Thetransmembrane protein Patched is a receptor for the morphogene SonicHedgehog. This protein associates with the smoothened protein totransduce hedgehog signals. SEQ ID NO:620 might represent a novel memberof the Patched family. Expression of SEQ ID NO:620 in normal andcancerous tissues was quantified by real-time RT-PCR usingsequence-specific oligos (SEQ ID NO:622, 623); see FIG. 27. SEQ IDNO:620 is highly expressed in lung. Compared to other normal tissues,SEQ ID NO:620 is overexpressed in ovarian cancer and melanoma. Based onthese expression results, SEQ ID NO:620 and its expression productsqualify as molecular markers and/or target candidates for targetedtherapies, in particular for these particular tumor types.

The nucleotide sequence according to SEQ ID NO:624 was deduced from SEQID NO:184 and codes for a 323 aa protein (SEQ ID NO:625) similar toTWIK-related acid-sensitive K⁺ channel, a member of the superfamily ofpotassium channel proteins that contain two pore-forming P domains.Expression of SEQ ID NO:624 in normal and cancerous tissues wasquantified by real-time RT-PCR using sequence-specific oligos (SEQ IDNO:626, 627); see FIG. 28. SEQ ID NO:624 is highly expressed in lung.Compared to other normal tissues, SEQ ID NO:624 is overexpressed ingastric cancer and lung cancer. Based on these expression results, SEQID NO:624 and its expression products qualify as molecular markersand/or target candidates for targeted therapies, in particular for theseparticular tumor types.

The presently described technology is now described in such full, clear,concise and exact terms as to enable any person skilled in the art towhich it pertains, to practice the same. It is to be understood that theforegoing describes preferred embodiments of the technology and thatmodifications may be made therein without departing from the spirit orscope of the invention as set forth in the appended claims.

1. A pharmaceutical composition, comprising an agent which (I) inhibitsexpression or activity of a tumor-associated antigen and/or (II) hastumor-inhibiting activity, and is selective for cells expressing orabnormally expressing a tumor-associated antigen and/or (III) whenadministered, selectively increases the amount of complexes between anMHC molecule and a tumor-associated antigen or a part thereof, saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from the group consisting of: (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574,577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and624, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c).
 2. The pharmaceutical composition as claimed in claim1, in which the agent under (II) causes induction of cell death,reduction in cell growth, damage to the cell membrane or secretion ofcytokines.
 3. The pharmaceutical composition as claimed in claim 1, inwhich the agent under (I) or (II) is an antisense nucleic acid whichhybridizes selectively with the nucleic acid coding for thetumor-associated antigen.
 4. The pharmaceutical composition as claimedin claim 1, in which the agent under (I) or (II) is an antibody whichbinds selectively to the tumor-associated antigen.
 5. The pharmaceuticalcomposition as claimed in claim 1, in which the agent comprises one ormore components selected from the group consisting of: (i) thetumor-associated antigen or a part thereof, (ii) a nucleic acid whichcodes for the tumor-associated antigen or a part thereof, (iii) anantibody which binds to the tumor-associated antigen or a part thereof,(iv) an antisense nucleic acid which hybridizes specifically with anucleic acid coding for the tumor-associated antigen, (v) an siRNAdirected against a nucleic acid coding for the tumor-associated antigen,(vi) a host cell which expresses the tumor-associated antigen or a partthereof, and (vii) isolated complexes between the tumor-associatedantigen or a part thereof and an MHC molecule.
 6. The pharmaceuticalcomposition as claimed in claim 1, in which the agent comprises two ormore agents which in each case selectively inhibit expression oractivity of different tumor-associated antigens, which are in each caseselective for cells expressing or abnormally expressing differenttumor-associated antigens or which increase the amount of complexesbetween MHC molecules and different tumor-associated antigens or partsthereof, with at least one of said tumor-associated antigens having asequence encoded by a nucleic acid which is selected from the groupconsisting of: (a) a nucleic acid which comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 541, 1-540,545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591,595, 599, 602, 606, 610, 613, 617, 620, and 624, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c).
 7. Apharmaceutical composition, comprising one or more components selectedfrom the group consisting of: (i) a tumor-associated antigen or a partthereof, (ii) a nucleic acid which codes for a tumor-associated antigenor a part thereof, (iii) an antibody which binds to a tumor-associatedantigen or a part thereof, (iv) an antisense nucleic acid whichhybridizes specifically with a nucleic acid coding for atumor-associated antigen, (v) an siRNA directed against a nucleic acidcoding for a tumor-associated antigen, (vi) a host cell which expressesa tumor-associated antigen or a part thereof, and (vii) isolatedcomplexes between a tumor-associated antigen or a part thereof and anMHC molecule, said tumor-associated antigen having a sequence encoded bya nucleic acid which is selected from the group consisting of: (a) anucleic acid which comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560,563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606, 610,613, 617, 620, and 624, a part or derivative thereof, (b) a nucleic acidwhich hybridizes with the nucleic acid of (a) under stringentconditions, (c) a nucleic acid which is degenerate with respect to thenucleic acid of (a) or (b), and (d) a nucleic acid which iscomplementary to the nucleic acid of (a), (b) or (c).
 8. Thepharmaceutical composition as claimed in claim 5 or 7, in which thenucleic acid of (ii) is present in an expression vector.
 9. Thepharmaceutical composition as claimed in claim 5 or 7, in which the hostcell secretes the tumor-associated antigen or the part thereof.
 10. Thepharmaceutical composition as claimed in claim 5 or 7, in which the hostcell additionally expresses an MHC molecule which binds to thetumor-associated antigen or the part thereof.
 11. The pharmaceuticalcomposition as claimed in claim 10, in which the host cell expresses theMHC molecule and/or the tumor-associated antigen or the part thereof ina recombinant manner.
 12. The pharmaceutical composition as claimed inclaim 10, in which the host cell expresses the MHC moleculeendogenously.
 13. The pharmaceutical composition as claimed in claim 5,7, 10 or 12, in which the host cell is an antigen-presenting cell. 14.The pharmaceutical composition as claimed in claim 4, 5 or 7, in whichthe antibody is a monoclonal, chimeric or humanized antibody, or is afragment of an antibody.
 15. The pharmaceutical composition as claimedin claim 4, 5, 7, or 14 in which the antibody is coupled to atherapeutic or diagnostic agent.
 16. The pharmaceutical composition asclaimed in any of claims 1-15, which may be used for the treatment orprevention of cancer.
 17. The pharmaceutical composition as claimed inclaim 16, in which the cancer is a lung tumor, a breast tumor, aprostate tumor, a melanoma, a colon tumor, a gastric tumor, a pancreatictumor, an ENT tumor, an ovarian tumor, a colorectal tumor, a cervicalcarcinoma, a colon carcinoma or a mammary carcinoma.
 18. Thepharmaceutical composition as claimed in any of claims 1, 2, 5-13, 16and 17, which is in the form of a vaccine.
 19. The pharmaceuticalcomposition as claimed in claim 18 for therapeutic and/or prophylacticuse.
 20. A method of diagnosing or monitoring a cancer disease, whichmethod comprises detecting or determining the quantity (i) of atumor-associated nucleic acid or of a part thereof, and/or (ii) of atumor-associated antigen or of a part thereof, and/or (iii) of anantibody to the tumor-associated antigen or a part thereof and/or (iv)of T lymphocytes which are specific to the tumor-associated antigen orto a part thereof in a biological sample isolated from a patient, saidtumor-associated nucleic acid being selected from the group consistingof: (a) a nucleic acid which comprises a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 541, 1-540, 545, 549, 553, 557,560, 563, 566, 570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606,610, 613, 617, 620, and 624, a part or derivative thereof, (b) a nucleicacid which hybridizes with the nucleic acid of (a) under stringentconditions, (c) a nucleic acid which is degenerate with respect to thenucleic acid of (a) or (b), and (d) a nucleic acid which iscomplementary to the nucleic acid of (a), (b) or (c), and saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from said group of nucleic acids.
 21. The method asclaimed in claim 20, in which the detection or determination of thequantity comprises (i) contacting the biological sample with an agentwhich binds specifically to the tumor-associated nucleic acid or to thepart thereof, to the tumor-associated antigen or the part thereof, tothe antibody or to the T lymphocytes, and (ii) detecting the formationof or determining the quantity of a complex between the agent and thenucleic acid or the part thereof, the tumor-associated antigen or thepart thereof, the antibody or the T lymphocytes.
 22. The method asclaimed in claim 21, in which the agent which binds specifically to thetumor-associated nucleic acid or to the part thereof is anoligonucleotide or polynucleotide, which hybridizes specifically to saidnucleic acid or to said part thereof.
 23. The method as claimed in claim21, in which the agent which binds specifically to the tumor-associatedantigen or the part thereof is an antibody binding specifically to saidtumor-associated antigen or to said part thereof.
 24. The method asclaimed in claim 21, in which the agent which binds specifically to theantibody is a protein or peptide binding specifically to said antibody.25. The method as claimed in claim 21, in which the agent which bindsspecifically to the T lymphocytes is a cell presenting the complexbetween the tumor-associated antigen or the part thereof and an MHCmolecule.
 26. The method as claimed in any of claims 20 to 25 whereinsaid monitoring of said disease comprises determining regression, courseor onset of said disease in a sample from a patient who has said diseaseor is suspected of falling ill with said disease.
 27. The method asclaimed in claim 26, which comprises a detection or determination of thequantity in a first sample at a first point in time and in a furthersample at a second point in time and a comparison of the two samples.28. The method as claimed in any of claims 21-27, in which the agent islabeled in a detectable manner.
 29. The method as claimed in any ofclaims 20-28, in which the sample comprises body fluid and/or bodytissue.
 30. The method as claimed in any of claims 20-29, in which saidcancer disease is characterized by expression or abnormal expression ofsaid tumor-associated nucleic acid.
 31. The method as claimed in claim30, in which said cancer disease is further characterized by expressionor abnormal expression of a tumor-associated antigen encoded by saidtumor-associated nucleic acid.
 32. A method of treating or preventing adisease characterized by expression or abnormal expression of atumor-associated antigen, which method comprises administration of apharmaceutical composition as claimed in any of claims 1-19, saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from the group consisting of: (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574,577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and624, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c).
 33. A method of treating, preventing, diagnosing ormonitoring a disease characterized by expression or abnormal expressionof a tumor-associated antigen, which method comprises administering anantibody binding to said tumor-associated antigen or to a part thereofand coupled to a therapeutic or diagnostic agent, said tumor-associatedantigen having a sequence encoded by a nucleic acid which is selectedfrom the group consisting of: (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583,587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and 624, a part orderivative thereof, (b) a nucleic acid which hybridizes with the nucleicacid of (a) under stringent conditions, (c) a nucleic acid which isdegenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c).
 34. The method as claimed in claim 23 or 33, in which the antibodyis a monoclonal, chimeric or humanized antibody, or is a fragment of anantibody.
 35. A method of treating a patient having a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen, which method comprises: (i) providing a sample containingimmunoreactive cells, (ii) contacting said sample with a host cellexpressing said tumor-associated antigen or a part thereof, underconditions which favor production of cytolytic or cytokine-releasing Tcells against said tumor-associated antigen or said part thereof, and(iii) introducing the cytolytic or cytokine-releasing T cells into thepatient in an amount suitable for lysing cells expressing thetumor-associated antigen or a part thereof, said tumor-associatedantigen having a sequence encoded by a nucleic acid which is selectedfrom the group consisting of: (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:541, 1-540, 545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583,587, 591, 595, 599, 602, 606, 610, 613, 617, 620, and 624, a part orderivative thereof, (b) a nucleic acid which hybridizes with the nucleicacid of (a) under stringent conditions, (c) a nucleic acid which isdegenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c).
 36. The method as claimed in claim 35, in which the host cellrecombinantly expresses an MHC molecule binding to the tumor-associatedantigen or to a part thereof.
 37. The method as claimed in claim 35, inwhich the host cell endogenously expresses an MHC molecule binding tothe tumor-associated antigen or to a part thereof.
 38. A method ofinhibiting the development of cancer in a patient, which methodcomprises administering an effective amount of a pharmaceuticalcomposition as claimed in any of claims 1-19.
 39. An agent, which bindsspecifically to a protein or polypeptide or to a part thereof, saidprotein or polypeptide being encoded by a nucleic acid selected from thegroup consisting of: (a) a nucleic acid which comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 541, 1-540,545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591,595, 599, 602, 606, 610, 613, 617, 620, and 624, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c).
 40. Theagent as claimed in claim 39, which is an antibody.
 41. The agent asclaimed in claim 40, in which the antibody is a monoclonal, chimeric orhumanized antibody, or is a fragment of an antibody.
 42. An antibody,which binds selectively to a complex of: (i) a protein or polypeptide ora part thereof and (ii) an MHC molecule to which said protein orpolypeptide or said part thereof binds, with said antibody not bindingto (i) or (ii) alone and said protein or polypeptide being encoded by anucleic acid selected from the group consisting of: (a) a nucleic acidwhich comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 541, 1-540, 545, 549, 553, 557, 560, 563, 566,570, 574, 577, 580, 583, 587, 591, 595, 599, 602, 606, 610, 613, 617,620, and 624, a part or derivative thereof, (b) a nucleic acid whichhybridizes with the nucleic acid of (a) under stringent conditions, (c)a nucleic acid which is degenerate with respect to the nucleic acid of(a) or (b), and (d) a nucleic acid which is complementary to the nucleicacid of (a), (b) or (c).
 43. The antibody as claimed in claim 42, whichis a monoclonal, chimeric or humanized antibody, or is a fragment of anantibody.
 44. A conjugate between an agent as claimed in any of claims39-41 or an antibody as claimed in any of claims 42-43 and a therapeuticor diagnostic agent.
 45. The conjugate as claimed in claim 44, in whichthe therapeutic or diagnostic agent is a toxin.
 46. A kit for detectingcancer, which kit comprises agents for detecting or determining thequantity (i) of a tumor-associated nucleic acid or of a part thereof,and/or (ii) of a tumor-associated antigen or of a part thereof, and/or(iii) of antibodies which bind to the tumor-associated antigen or to apart thereof, and/or (iv) of T cells which are specific for a complexbetween the tumor-associated antigen or a part thereof and an MHCmolecule, said tumor-associated nucleic acid being selected from thegroup consisting of: (a) a nucleic acid which comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 541, 1-540,545, 549, 553, 557, 560, 563, 566, 570, 574, 577, 580, 583, 587, 591,595, 599, 602, 606, 610, 613, 617, 620, and 624, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c), and saidtumor-associated antigen having a sequence encoded by a nucleic acidwhich is selected from said group of nucleic acids.
 47. Thepharmaceutical composition as claimed in any of claims 1-19 or themethod as claimed in any of claims 20-38 in which the tumor-associatedantigen comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 542, 546, 550, 554, 567, 571, 584, 588, 592,596, 603, 607, 614, 621, and 625, a part or derivative thereof.
 48. Theagent as claimed in any of claims 39-41, the antibody as claimed in anyof claims 42-43 or the conjugate as claimed in any of claims 44-45 inwhich the protein or polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 542, 546, 550, 554,567, 571, 584, 588, 592, 596, 603, 607, 614, 621, and 625, a part orderivative thereof.